Peptide

For education only

This guide cannot diagnose a condition or recommend a personal treatment plan. Discuss medical questions with a qualified professional.

Chapters and Articles

You might find these chapters and articles relevant to this topic.

Peptides

Peptides are versatile molecules with high cosmeceutical interest, they have been developed in response to different skin conditions in the field of cosmeceuticals. A wide range of these peptides target skin aging, regulating mainly collagen turnover, and blocking or promoting specific neurotransmitters, aiming decreasing age-induced wrinkles. Many of these peptides are synthetic peptide-based categorized as neurotransmitter inhibitor peptides, carrier peptides, and signal peptides. All these classes were well-reviewed by Errante et al. (Errante et al., 2020). Nevertheless, peptide-based molecules can be also obtained from vertebrate and invertebrate animals, plants, bacteria, fungi, and protozoans with distinct biological bioactivities. Recently, more attention has been given to agro-industrial by-products and residues comprising significant amounts of protein (10 – 50%) such as rice bran, coconut pulp, and soybean meal which are excellent and promising sources of bioactive peptides (Gorguc et al., 2020). The biological activities of peptides will also diverge according to molecular weight, sequence, and type of amino acids. In the literature, a range of bioactivities is described, including antioxidant, antihypertensive, antimicrobial, and antidiabetic, among others (Freitas et al., 2019; Karami and Akbari-Adergani, 2019; Piovesana et al., 2018). In the skin, they also have been used to treat pigmentation, improve extracellular matrix synthesis, and modulate innate immunity and inflammation. The use of peptides has some advantages such as their selectivity, the absence of premarket regulatory requirements for their use, and their lack of immunogenicity. In contrast, the low lipophilicity and high molecular weight of peptides can negatively affect their absorption in the skin. Therefore, more clinical evidence must prove the efficacy of peptides for dermatological uses (Pai et al., 2017).

Antimicrobial peptides

Antimicrobial peptides (AMPs) are a specific type of peptides, they are normally small (12–50 amino acids), positively charged amphiphilic molecules with α-helix or β-sheet linear motifs, and linear or cyclic configurations. The most frequent applications of AMPs include kill or inhibit the microbial growth and protect the skin from infections against bacteria, yeast, fungi, protozoa, and viruses (Liu et al., 2013; Pai et al., 2017). It is important to note that AMPs can be found in all living animals (from microorganisms to mammals) as part of their primary mechanisms used in the early stages of immune defense. The human skin can equally secrete them onto the skin surface, while others are expressed in the healthy skin but only upregulated in specific conditions such as the presence of microorganisms, proinflammatory cytokines, chemokines, radiation, wound healing, etc. (Herman and Herman, 2019; Miazga-Karska et al., 2020). Deregulations on AMPs secretion by the skin contribute to the pathogenesis of several skin diseases such as atopic dermatitis, acne vulgaris, and psoriasis (Mangoni et al., 2016). Nowadays, with the emergence of bacterial resistance, nature-derived AMPs can be a viable alternative to classic antibiotics when the immune system is unable to deal with the infection. These positively charged AMPs interact with the negatively charged bacterial cell membranes without causing any damage to Eukaryotic membranes. The most frequently described mechanism of action of AMPs is the disruption of membrane potential that ultimately caused cell death (Hammami et al., 2009). Other AMPs can translocate across the bacterial membranes without any detrimental effects on themselves but destabilize other cell functions (including inhibition of nucleic acids, proteins, or cell wall synthesis).

A great number of AMPs have been isolated from plant species, including roots, seeds, flowers, and leaves. The high content of thionins and defensins (cysteine residues) in plants produces two to six disulfide bonds that eventually will affect resistance to proteolytic degradation (Mangoni et al., 2016; Miazga-Karska et al., 2020). Compared with other kingdoms, plant-derived AMPs have larger diversity and abundance due to probably environmental evolutionary forces and redundant genomes. Every single plant can be an arsenal of AMPs for the most diversity of pathogens. A recent publication by Petre (2020) discusses the concept of host-defense peptides (HDPs), which are peptides with both antimicrobial and immunomodulatory activities. This concept arises from the ability of peptides to change the physiology of the plant and trigger a set of immunomodulatory mechanisms. However, no HDPs were reported in plants so far, as the concept of HDP has not been taken yet by the plant science community (Petre, 2020). Nevertheless, some progress has been achieved in AMPs for the treatment of skin infections. Propionibacterium acnes is a gram-positive human skin commensal, one of the most frequently found that makes difficult the treatment of acne. Miazga-Karska et al., 2020 isolated low molecular weight peptides from burdock roots (Arctium lappa l.) and assessed the level of antibacterial activity against Gram-positive and Gram-negative bacteria. Both tested peptides (Br-f and Br-p) only inhibited the growth of Gram-positive bacteria without cytotoxic effects in skin fibroblast cultures. More precisely, Br-p had bactericidal nature against all Gram-positive strains, while Br-f was only active against anaerobic Gram-positive strains. These properties of active peptides have allowed them to be used to produce therapeutic dressing material (Br-p/chitosan/sodium alginate) to be used in infections against Gram-positive bacteria in acne skin. These specificities allowed the development of a therapeutic dressing material (Br-p/chitosan/sodium alginate) to be used in infections against Gram-positive bacteria (Miazga-Karska et al., 2020). Indeed, the AMPs have revealed promising results against diverse acne pathogens, including also Staphylococcus epidermidis, Staphylococcus aureus, and Candida albicans strains (Ma et al., 2022; Theansungnoen et al., 2022). The following two ferns, Adiantum Edgeworth and Adiantum capilllus-veneris from the Himalayas have been explored for the presence of AMPs with antimicrobial activity for different pathogens, including Staphylococcus aureus. Proteins from both ferns showed antibacterial activity against this bacterium and the experiments confirmed the presence of AMPs. In this work, the isolation, characterization, and sequencing of purified AMPs were not performed, but the researchers highlighted the importance of addressing these experiments (Negi and Maurya, 2020). Other AMPs have shown their efficacy against Staphylococcus aureus, mainly in cases where there is resistance to conventional treatments. Pardaxin (GE33) is a peptide isolated from Red Sea flatfish (Pardachirus marmoratus) with antimicrobial activity against both Gram-positive and Gram-negative bacteria. Like other antimicrobial peptides, Pardaxin disrupts bacterial membranes, but it also stimulates the arachidonic acid cascade to affect the extracellular-signal-regulated kinase (ERK) and other signaling pathways in treated cells (Bloch-Shilderman et al., 2001). Huang et al. (2014) evaluated the suitability of this AMP as a wound-healing agent in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) infection and obtained the same consistent results. Pardaxin demonstrated to have antibacterial activity against MRSA in vitro and decreased MRSA-induced TNF-α at the wound site. The data also showed the increased recruitment of macrophages and monocytes to the site of infection, and the stimulation of signaling pathways responsible for the induction of specific chemokines. In mice treated with Pardaxin, the reepithelialization and dermal maturation were also faster than in mice treated with vancomycin. The untreated mice and mice treated with methicillin died, but in contrast, mice treated with pardaxin survived. The authors support the idea that Pardaxin may be suitable for conditions in which there is a high risk of infection once it showed prophylactic efficacy. Otherwise, this AMP is compatible with the use of several antibiotics and does not display any apparent immunotoxic effects (Huang et al., 2014). Similar results were obtained by Cunsolo et al. (2020) with novel AMPs from Charybdis pankration, a Mediterranean plant, well-known for its biological properties in traditional medicine. Polypeptide-enriched extracts from different parts of the plant were tested against two relevant pathogens, Staphylococcus aureus, and Pseudomonas aeruginosa. The critical antibiotic resistance of these two pathogens has increased the concerns about their ample diffusion in natural environments. They are the most recurrent causes of wound-associated infections (diabetic foot, venous leg, and pressure ulcers). In this study two peptides were identified to be good candidates as AMPs themselves, but also as chemical platforms to develop efficient antimicrobial drugs due to their physicochemical parameters, including their positive charge, the presence of hydrophobic residues, and their stability, which make them potentially able to approach and interact with bacterial membranes (Cunsolo et al., 2020).

In summary, several AMPs derived from plants have revealed antimicrobial activity against both Gram-positive and Gram-negative strains, but their action is not restricted to pathogens. They also can modulate several immune mechanisms related to inflammation and wound healing in the host. Therefore, more studies should be performed to understand their applications on the skin, as well as their safety for topical use (Moyer et al., 2021; Taniguchi et al., 2017).

Nature-derived peptides

Nowadays, innovation in cosmetics is based on new bioactive formulations such as oils, vitamins, protein hydrolysates, and peptides. Several amino acids are already included in skincare products mainly due to their ability to act as water-binding molecules. More than that, there is a growing interest in bioactive peptides for stimulating collagen and elastin synthesis in the skin. Although most peptides in cosmetics are of synthetic origin, natural peptide extracts represent a competitive alternative, not only because of the need to explore new natural products but also by their multifunctionality. The protein source, amino acid composition of peptides, enzyme selection for hydrolysis, peptide enrichment method, as well as stability of the peptides within the formulations, and bioavailability will be decisive for the biological activities observed in skin cells (Ahsan, 2019; Skibska and Perlikowska, 2021).

Marine sources are an excellent source of bioactive peptides for the most diverse applications, including cosmetic industries. The biological and functional properties of peptides derived from marine sources are already well-reviewed by Cunha and Pintado (2022) highlighting the versatility of these molecules (Cunha and Pintado, 2021). Many of these peptides have been isolated from algae that can be used in cosmetic formulations with different purposes, such as anti-aging creams, refreshing care products, depigmentation products, or emollients. Different from other species in marine biomass is relatively easier to abstain from higher amounts of protein from algae and consequently isolate and identify higher amounts of peptides. Peptides derived from algae are already used in cosmetic formulations such as the Dermochlorella® (isolated from Chlorella Vulgaris) that promises the stimulation of collagen synthesis (Martins et al., 2014). In fact, the genus Chlorella and Arthrospira represent the most recognized sources of molecules in the skincare market. The Chlorella-derived peptide (CDP) fraction has shown the ability to decrease the gene expression of several proteins in UVB irradiated fibroblasts, such as MMP-1 and cysteine-rich 61 (CYR61), and monocyte chemoattractant protein-1 (MCP-1). The activation of the MMPs is associated with photoaging accelerating the degradation of skin collagen and inhibiting the collagen synthesis of ECM. The inhibition of MMP expression or activation of collagen synthesis may be a strategy to prevent wrinkle formation associated with photoaging (Chen et al., 2011). In the same way, peptide-enriched extracts from the microalga Arthrospira platensis were effective in promoting skin hydration, increasing the gene expression of several factors specifically involved in the water balance maintenance in keratinocytes (aquaporin3, hyaluronic acid synthase 3, and filaggrin). In addition, it was observed the inhibition of ROS production was caused by oxidative stress agents (Chien et al., 2013).

Several plant-derived peptides have shown their efficacy as anti-aging promoters in skin formulations, mainly in skin creams of personal care. Peptides that contain similar amino acid sequences to the ECM proteins present in the human skin have a great potential to stimulate dermal ECM synthesis, stimulating re-epithelialization, promoting cell adhesion, and supporting tissue regeneration. Glycine max (soy) lysate has shown these properties, being considered with excellent wound healing properties (Apone et al., 2019; Chien et al., 2013). Lotus japonicus have also in their composition key amino acids that will promote the renovation of some ECM components, including collagens and periostin (Tito et al., 2019).

Some limitations of nature-derived peptides must be considered such as the interactions with other unspecific biological components and the risk of potential allergens. On the other way, the isolation and characterization of a single peptide faction may be very challenging with elevated costs. For these reasons working with synthetic peptides became sometimes more attractive. To avoid these problems, the peptides can be derived from plant tissue cultures, that are grown in the laboratory, under controlled and axenic conditions. Different from peptides derived from plant extracts, these ones decrease the risk of the presence of potential allergens, and the environmental pollutants are almost completely abolished (Guzmán et al., 2007; Jack et al., 2013).

Read full article

URL: https://www.sciencedirect.com/science/article/pii/S094471132300185X

Peptides

Peptides are compounds of great medical interest due to their physiological role as hormones and neurotransmitters. Furthermore, considering peptides as subunits of proteins, peptide mapping after chemical or enzymatical cleavage allows characterization of the protein and to reveal metabolic disorders.

Physicochemical Nature of Peptides

The characteristics of peptides are situated between those of amino acids and high molecular weight proteins. Oligopeptides containing up to 15 amino acids behave similarly to amino acids. Short peptide chains cannot create a complicated conformation. In contrast, very long polypeptides with chain lengths up to approximately 100 units behave like small proteins. They exhibit characteristic features of secondary and tertiary structure.

Peptides exist in aqueous solution as amphoteric ions. Therefore peptides possess isoelectric points (pI). The peptide has net electroneutral properties at the pI. The zwitterionic characteristics are influenced predominantly by the acidity of the medium. In acidic media the carboxyl group (pKa∼2.7–2.9) is protonated and the peptide behaves as a cation. In alkaline media the protonated ammonium group is eliminated and the zwitterionic form is converted into an anion. The degree of dissociation is determined by the dissociation constants of the functional groups yielding different net charges.

A further feature to be considered in electrophoretic behaviour is the sequence of the amino acids. The dissociation constants of the individual residues are affected by the arrangement of the amino acids in the chain. Mass-to-charge ratios are altered and the peptide exhibits a different mobility.

Prediction of Electrophoretic Mobility of Peptides

A theoretical model of electrophoretic migration underlying the experimental approach can be very useful for the optimization of analytical conditions. It supports predicting peptide mobilities under different experimental conditions such as pH. If selectivity between closely migrating species has to be implemented, the model facilitates adjustment of the separation environment.

Furthermore, considering technical processes and purity control of peptide synthesis or enzymatic digestion of proteins, a defined relationship between apparent mobility and physicochemical parameters supports the identification of unknown species. Variation in the sequence of peptides can also be easily determined.

The mathematical description of the migration process is based on the contribution of two forces. The electrical field accelerates an ion with a force proportional to its charge. In addition, the ion is influenced by a retarding force which results from the viscosity of the medium and is connected to a size parameter.

For permanently ionized small ions a prediction of migration is easily achieved by applying Stoke's law which correlates mobility with qr−1 and qM−1/3 (q, charge and M, molecular mass). With larger, more complex aggregates like peptides both the charge and a suitable size parameter has to be ascertained. For computing the charge, the sequence of amino acids has to be considered as the environment of a residue affects the extent of ionization, e.g. neighbouring amide bond or acidic/basic residues at terminal amino or carboxylic groups. This means that the ionization constants of the free amino acids have to be adjusted. During the development of a theoretical understanding of migration phenomena, many approaches have been made considering mass, surface, radius and the number of units in a peptide chain as size parameter.

The following equation results from the semi-empirical approach by Offord relating electrophoretic mobility μ of peptides with their charge q and their molecular mass M:

μ=k×q×m−2/3

This linear relationship has been validated by experimental research; a large set of analytes covering collagen fragments, tryptic digest of human growth hormone (33 peptides), motilin fragments (24 peptides) and many additional peptides differ widely in charge and amino acid sequence. Nowadays several computer programs are available which are capable of calculating the charge-to-mass ratio just requiring the amino acid sequence.

Electrophoretic Systems – Separation Strategies

To optimize the separation of peptides, the experimental conditions have to be adjusted to emphasize differences in the charge-to-mass ratios of the analytes.

Apart from external parameters like electrical field, capillary dimensions (length, inner diameter) and temperature, separation is mostly influenced by the electrolyte. Intrinsic variables like type of buffer, mobility, ionic strength, pH and buffer additives determine electrophoretic and electroosmotic mobility.

In the first place selectivity in the analysis of peptides is controlled by pH. Altering the acidity of the separation medium affects both the charge of the peptide and the ionization of the capillary wall, resulting in the change in EOF.

The hysteresis-like course of the EOF shows the greatest variation in the pH range of approximately 5–7, i.e. near the dissociation constant of the silanol groups. For pH values below 3 or greater than 9, the influence of the superimposed EOF can be neglected and the migration of the peptide is almost independent from the EOF.

In acidic media (pH∼2) both basic and acidic residues of the peptide are protonated. Selectivity is attributed to the number of positive-charged ammonium groups in the chain resulting in different charge densities. Analytes migrate with the EOF. In high pH buffers (pH∼10), deprotonation of terminal and side chain ammonium groups (His) induces negatively charged species (presence of carboxylate groups) which migrate in the opposite direction to the EOF. At higher pH values the side chain amino groups of arg and lys are the only ones affected.

Optimization of pH values below 2 and above 12 is difficult to achieve since the limiting values of mobility are reached. Furthermore, due to the high conductivities of protons and hydroxyl ions, high currents accompanied by Joule heating are generated. For practical purposes selectivity control for peptides with a majority of acidic moieties is mainly achieved in the range of pH 3–6 while basic residues are mostly affected at pHs around 10.

Additionally, isoelectric points of the peptides have to be included in the optimization strategy.

If peptides are obtained by chemical or enzymatic digests of proteins the cleaving agent has to be considered, e.g. trypsin cuts at the C-terminal side of lys and arg respectively. Thus fragments contain an excess of acidic residues. Selectivity can be easily affected in acidic media. Cleavage at aromatic or aliphatic side chains is performed with chymotrypsin or pepsin, yielding fragments with both acidic and basic residues and optimization can be extended to the full pH range (Figure 6).

Figure 6. Tryptic digest of a haemoglobin variant separated by CZE. Capillary: poly(vinyl alcohol) coated fused silica capillary 50 μm i.d., 50/57 cm, buffer: phosphate 50 mmol L−1; pH = 2.5; E = 526 V cm−1, 214 nm; injection 0.5 psi, 5 s.

Frequently used electrolytes for peptide mapping are phosphate, citrate and acetate as acidic buffers while borate or TRIS/Tricine are mainly applied under basic conditions. Phosphate and citrate are buffers that can be used over a broad pH range due to their multiple association constants. Borate exhibits very low conductivity compared to phosphate and other buffers. Buffer concentrations in the range of 10 mmol L−1 to approximately 100 mmol L−1 can be used. The electrolytes used should not possess any UV absorbance at low wavelengths.

An increase in ionic strength generates sharper peaks (zone focusing) due to the drop of the electrical field at the sample–electrolyte boundary and sample loading capacity can be increased. High ionic strengths induce high electrical currents and the increase of Joule heating can give rise to band broadening.

Dispersive effects caused by the interaction with the capillary wall are usually not a problem with peptides but larger species can exhibit characteristics similar to proteins in that they tend to adsorb at the capillary wall.

High ionic strength, extreme pH values and buffer additives competing in adsorption with the peptides are strategies of optimization which can be adapted from protein analysis. At extreme pH values, peptides and the capillary wall are equally charged so electrostatic repulsion diminishes adsorption. Coated capillaries have been used to suppress this phenomenon.

High salt content in the sample may destroy the separation efficiency of the electrophoretic system so sample preparation steps must remove the high ionic strength in the sample.

Enhancement in selectivity can be attained if an additional equilibrium is superimposed on to the electrophoretic process. Mostly the additives used for this are complexing agents which interact with specific groups of the peptide.

As for amino acids, metal ions can be employed for the separation of peptides and histidine-containing peptides especially interact with zinc salts. Separation of two histidine dipeptides (l-l, d-l) can be attributed to favourable steric arrangement of the histidine residues in one isomer.

Cyclodextrins form dynamic inclusion complexes with hydrophobic parts of the peptide, e.g. with amino acid residues containing aromatic rings like phenylalanine. The mass of a complexed analyte is increased in this way and lower charge-to-mass ratio results in decreased mobility.

Ion-pairing reagents like short chain alkylsulfonic acids are particularly applied to adjust selectivity for hydrophobic peptides. Concentrations below the critical micellar concentration are used. The mechanism is based on the interaction between the hydrophobic surface of the peptides and the hydrophobic alkyl chain. Depending on the hydrophobicity of a peptide, different amounts of alkylsulfonic acid are attracted. Charge-to-mass ratios of the individual peptides are influenced to a different extent leading to the separation of the species.

A second approach to impart selectivity to large peptides with identical mobilities but different hydrophobicities is the use of ion-pairing reagents above their critical micellar concentration (CMC). This technique may also be used for peptides differing in neutral amino acids such as ala, val, leu or ile. MEKC takes advantage of the partitioning of the peptides between the electrolyte and the pseudo-stationary phase of the micelles. Hydrophilic moieties of the peptide interact with the outer polar sections of the micelle whereas hydrophobic parts are situated in the inner hydrophobic sphere. These peptide–micelle aggregates possess a different mobility compared to the electrophoretic mobility of the peptide in free solution.

Types of surfactants employed are divided into anionic, cationic and nonionic micelle-forming reagents. Because of the different charges, different migration directions are obtained. Negatively charged SDS, one of the most frequently used additives, migrates counter to the EOF and is used in concentrations up to approximately 150 mmol L−1.

Common positively charged reagents are cetyl, dodecyl and hexadecyltrimethylammonium salts. These reagents invert the EOF at concentrations below the CMC so that as a consequence the polarity of the applied electrical field has to be reversed.

The addition of organic solvents such as methanol, ethanol, acetonitrile or tetrahydrofuran can provide selectivity for closely migrating peptides. These changes can be mainly attributed to solvation of side chains and variations in dissociation of the functional groups of the peptide. Additionally the EOF is modified due to altering the ζ- potential and the increase in buffer viscosity which generates a lower EOF and lower currents. In this way separations have been established for peptides differing in only a single neutral amino acid.

Peptides, especially large peptides with protein-like characteristics, sometimes tend to adsorb at the capillary wall. Beside the possibilities for avoiding dispersive effects mentioned above, the addition of amino- or diamino compounds like diamino-pentane, butane or morpholine can diminish the peptide–wall interaction. Competing equilibria in the electrostatic attraction between analyte-silanol and amine-silanol groups suppress the adsorption of the peptide. Another approach to reduce adsorption is derivatization of the silanol groups with an uncharged polymer (coated capillaries).

Detection Techniques

The detection of peptides suffers from the same difficulties as described for amino acids. Additionally only a few amino acids (phe, try, tyr and to a lesser extent his, arg, gln, asn) provide residues with strong chromophores.

Measuring UV absorbance at low wavelengths (<220 nm) is the commonest mode of detection to give limits of detection of about 1 μg mL−1 (∼10−5–10−6 mol L−1) which are sufficient for most applications. Spectra obtained by a photodiode array detection support identification of impurities in peptide synthesis due mainly to the absence of the characteristic absorbance of aromatic residues at 220 nm (Figure 7).

Figure 7. CZE separation of a peptide mixture. Capillary: ethylene/vinyl acetate dynamically coated with polyvinyl alcohol 75 μm i.d., 25/45 cm, buffer: phosphate 50 mmol L−1; pH = 2.5; E = 155 V cm−1, 200 nm; injection 50 mbar, 5 s. 1, Bradykinin; 2, angiotensin II; 3, α-MSH; 4, TRH; 5, LH-RH; 6, leucin enkephalin; 7, bombesin; 8, methionin; 9, oxytocin.

Indirect techniques can be applied as for amino acids.

Detection of trace amounts of peptides requires more sensitive methods and sensitivity can be improved by fluorescence methods.

This approach faces the same difficulties as UV absorbance detection in that only try and, to a lesser extent, tyr and phe exhibit native fluorescence when excited at 280 nm (Xe-lamp). However, this ‘natural specificity’ facilitates selective identification of try-containing peptides. In addition, indirect fluorescence detection using salicylic acid for anionic charge peptides (basic buffers) or quinine for the positive mode (acidic buffer) have been applied.

To accomplish lower detection limits for a broader range of species derivatization techniques have to be applied and all the agents described for amino acids can be used for the derivatization of peptides.

Increased interest is being paid to mass spectrometric techniques for the characterization of peptides, especially soft ionization techniques like electron spray ionization (ESI). A promising approach towards nonfragmented peptides is the matrix-assisted laser desorption ionization with time-of-flight mass spectrometers (MALDI-TOF).

Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B0122267702037819

1 Introduction

Peptides are a kind of compounds formed by linking α-amino acids through peptide bonds. For example, calcitonin, which can treat deformity osteitis and hypercalcemia, is composed of 32 amino acids [1]. Protein is composed of multiple peptide chains entangled in a certain spatial structure. For example, human insulin that can lower blood sugar has two peptide chains connected by two disulfide bonds, containing 16 types of 51 amino acids [2]. Polymers composed of less than 40 amino acids are considered as peptides by Food and Drug Administration (FDA) under the regulation of the Federal Food, Drug, and Cosmetic Act (FD&C Act) [3]. Peptide and protein drugs (PPDs) can effectively treat diabetes, osteoporosis, cancer, gene defects and other diseases [4]. They have high specificity and tolerance, low immunogenicity and other advantages. However, the difficulty in delivery and low bioavailability of PPDs greatly limit their clinical application [5].

PPDs are hydrophilic macromolecules with molecular weights ranging from 300 Da to greater than 1000 kDa [6]. According to the Lipinski's Rule of Five [7], PPDs are difficult to penetrate the lipophilic gastrointestinal mucosa. Unstable PPDs are susceptible to denaturation under the action of gastric acid [8], and then resulting in low bioavailability of oral delivery (less than 1%) [2,8]. Currently, most PPDs are administered by subcutaneous injection [7]. However, subcutaneous injection requires professional medical personnel to operate [9]. In addition, due to the short half-life of PPDs after subcutaneous administration, frequent injections are required, which will bring pain and psychological pressure to the patients and reduce the patient's compliance [2]. Therefore, the development of effective delivery technology for PPDs with less invasiveness, controllability, and high bioavailability is the main challenge in clinical applications.

Transdermal drug delivery (TDD) is a noninvasive or minimally invasive method that allows a certain amount of drug to pass through the epidermal layer of the skin by free diffusion or other means and continues to enter the systemic blood circulation at a controlled rate. TDD has the following advantages: 1) The large surface area of the skin (1–2 m2, accounting for 1/3 of the blood circulation) provides a larger accessible area for TDD. And the transdermal route can avoid gastrointestinal degradation and liver first-pass effect, thereby improving the drugs bioavailability [6]. 2) It can be administered at a certain rate for a long period of time (lasting several hours to several days) [2]. 3) Compared with subcutaneous injection, TDD has less trauma, lower risk of infection, lower cost, high patient compliance, and can achieve patient self-management [10]. 4) The skin has a dense network of antigen-presenting cells, which makes it a convenient route for vaccination to induce an immune response.

However, since the stratum corneum is formed by desmosome-connected keratinocytes embedded in a highly organized lipid layer (containing ceramide, fatty acids and cholesterol), it provides a natural physical and water-retaining barrier to protect the subcutaneous tissue from infection, dehydration, and external chemical or physical damage [11]. The key of the TDD technology is how to make the drug through the stratum corneum to reach the site of action. And there are three ways for drugs to pass through the stratum corneum [6]: 1) directly through keratinocytes and intercellular lipids (intracellular pathway); 2) through the gap between keratinocytes (intercellular pathway); and 3) through skin accessories including hair follicles and sebaceous glands. Among them, the intracellular pathway requires the partition of the drug into multiple hydrophilic and hydrophobic domains, which is detrimental to drug delivery. As the skin accessories only accounts for 1% of the total skin area, drugs are mainly transported through a tortuous path between cells [12]. Drug passive permeation across the stratum corneum follows Fick's first law (equation (1)):

(1)dmdt=J=DC0Ph

where steady-state flux (J) is related to the diffusion coefficient (D) of the drug in the stratum corneum over a diffusional path length or membrane thickness (h), the partition coefficient (P) between the stratum corneum and the vehicle, and the applied drug concentration (C0) which are assumed to be constant [12]. Therefore, the molecular weight of drugs that can be passively transported through the skin to less than 500 Da, its log P (octanol/water) is between 1 and 3, and the required dose is small [2]. By the end of 2017, there were only 54 transdermal patches (28 novel products and 26 generic products) for drugs in the market [13]. Among them, only PPDs were approved by FDA to delivery of inactivated influenza vaccines via TDD by hollow microneedles [14]. Furthermore, more than 20 PPDs for TDD are in clinical trials and about half of them have entered Phase II or III clinical studies. The majority of the current clinical trials (>15) are based on microneedles (MNs) or jet injection systems [15].

Transdermal patches can be divided into reservoir-type and matrix-type patches [16]. As shown in Fig. 1a, the reservoir-type patches allow the sustainable release of the drug in a solution or gel, and the delivery is governed by a rate-controlling membrane. These type of patches are mainly used in the research of active TDD systems such as iontophoresis and ultrasound. Although they can provide a constant drug release rate, their application has been significantly limited due to the drug leakage induced by manufacturing aberration of the patches [17]. Therefore, 88% of FDA-approved novel products and patches approved after 1999 are matrix-type patches [13]. Matrix-type patches, which were introduced in the 80s, incorporate drug in an adhesive (as shown in Fig. 1b) or polymer matrix, and the rate of drug delivery is controlled by skin permeability. Thus, matrix-type patches may maintain sustained steady-state drug level in the blood through manipulating the drug concentration and vehicle components in the patch and/or the area of skin exposed to the patch [17]. It is worth noting that the drug and excipient loaded in the patches may undergo phase changes (dissolved drug may crystallize, the dispersed drug may agglomerate), which are more likely to occur in matrix-type. Thus, when selecting a pressure-sensitive adhesive for the matrix-type patches, the following four points need to be considered: solubility of drug and excipients; effect of dissolved/dispersed additives on adhesion; long-term stability of dissolved/dispersed components; compatibility of the backing layer [18]. However, these two types of traditional patches strongly rely on the passive diffusion of drugs, which pose a serious challenge for the transdermal delivery of PPDs with poor stability, large molecular weight, or hydrophilicity [19].

Fig. 1. Reservoir-type (a) and matrix-type (b) transdermal delivery systems [16].

After decades of research, various strategies such as skin penetration enhancers, micro-/nanocarriers, iontophoresis, electroporation, jet injection, thermal ablation, and MNs have been developed for delivering hydrophilic PPDs to the systemic circulation. Fig. 2 briefly illustrates the basic principles of different technologies. Recently, many reviews about different transdermal delivery technologies of PPDs, including MNs [20], nanocarriers [21], penetrating peptides [22], have been published. In addition, there are also some reviews that introduce several techniques to enhance the transdermal delivery of PPDs. For example, Chaulagain et al. reviewed non-invasive techniques including enhancers, nanocarriers and certain biological peptides for their applications in protein delivery [7]. Hwang et al. also summarized the physical enhancement methods, including iontophoresis, electroporation, and ultrasound, to the transdermal delivery of protein drugs [23]. But there are few reviews that comprehensively summarize and compare different techniques for enhancing transdermal delivery of PPDs. And in this review, we summarize and evaluate the mechanism, advantages and disadvantages of different technical methods, hoping to provide scientific guidance for relevant researchers and enterprises. In addition, the mechanism of five latest technologies, including ionic liquids, STAR particles, temporal pressure, nanofiber patch, theranostic MNs, are also briefly introduced.

Fig. 2. Strategies to enhance the transdermal delivery of PPDs.

Read full article

URL: https://www.sciencedirect.com/science/article/pii/S177322472031296X

7.08.2 Peptides/Proteins

Peptides and proteins are linear polymers that are made up of monomers known as α-amino acids, whose chemical structure is shown in Figure 1(a). Proteins are much larger in length than peptides and describe complete, biological macromolecules with a stable conformation, whereas peptides are usually short amino acid oligomers. There are 20 natural amino acids, which are tabulated and classified in Figure 1(b) based on the chemical nature of their side chain. Each amino acid has a corresponding three- and one-letter code, which are listed in the table. The most important characteristic of an amino acid side chain is its polarity, which largely dictates protein folding. Nonpolar side chains, such as methyl groups, are hydrophobic and prefer not to interact with water, whereas polar side chains, such as carbonyl, amino, hydroxyl, and sulfhydryl groups, are hydrophilic and can interact with water. The amino acids can join together into long chains by forming amide bonds between the –NH2 group of one amino acid and the –COOH group of another. The long repetitive sequence of –NH–CH–CO– atoms that make up the protein constitutes the backbone, but the sequence of side chains, or the primary structure, ultimately determines the higher order secondary and tertiary structures (or three-dimensional (3D) structure) of the protein, which are critical to its function. The terms ‘primary’, ‘secondary’, and ‘tertiary’ structure emphasize the hierarchical nature of protein structure, which makes them unique in comparison to synthetic polymers.

Figure 1. (a) Chemical structure of α-amino acids. (b) Table listing the 20 natural amino acids categorized by the chemical nature of the side chain R group. (c) Representation of the dihedral angles in a peptide backbone.

7.08.2.1 Primary Structures

The primary structure refers to the sequence of the different amino acids in a peptide or protein. The two ends of the peptide chain are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus), based on the nature of the free group on each extremity. The primary structure is typically read from the N-terminus to the C-terminus, using the three- or one-letter amino acid codes. The primary structure is unique to a particular protein and ultimately defines the structure and function of that protein.

7.08.2.2 Secondary Structures

Secondary structure refers to the local, specific, geometrical shape of a peptide. The conformations of peptide backbones are restricted by steric clashes between backbone and side chain atoms. The allowed values for the backbone dihedral angles (φ, ψ), shown in Figure 1(c), define the spatial orientation of the peptide. This, coupled with the formation of hydrogen bonds between the NH and CO groups of the backbone, leads to regular secondary structures, such as the α-helix and the β-sheet, the two most predominant types of protein secondary structures.

7.08.2.2.1 α-Helix

The most common secondary structure found in natural proteins is the α-helix, shown in Figure 2(a). The α-helix is a right-handed coil with backbone dihedral angles of φ = –60° and ψ = –45°, in which each amino acid residue corresponds to a 100° turn in the helix and a rise of 1.5 Å along the helical axis, so that the helix has 3.6 residues per turn, and the backbone CO group of the ith residue forms a hydrogen bond with the NH group of the i + 4th residue. Thus, all NH and CO groups, except the N- and C-termini, are involved in hydrogen bonds. Helices observed in natural proteins can range from 4 to over 40 residues in length. The radius of the helix, excluding side chains, is 2.3 Å. The thermodynamically driven process of helix folding is a balance of the entropic cost associated with the folding or constraining of the peptide chains and the stabilizing effects of forming intrahydrogen bonds. Because all of the backbone amide groups are involved in intrachain hydrogen bonds, the interactions of helices with other peptides or molecules occur exclusively through side chain interactions. Amphipathic helices, in which one face of the helix is polar and the other is nonpolar, are common, thus allowing for the formation of higher order structures by intermolecular interactions, such as hydrophobic and electrostatic interactions, which will be discussed later.

Figure 2. (a) Structure of an α-helix that shows hydrogen bonds between the CO group of the ith residue and the NH group of the i + 4th residue. (b) Structures of parallel and antiparallel β-sheets that show the difference in interpeptide hydrogen bond patterns.

7.08.2.2.2 β-Sheet

The other common secondary structure is the β-sheet, shown in Figure 2(b). β-Sheets consist of two or more β-strands connected laterally by backbone hydrogen bonds, generally forming a twisted, pleated sheet, with dihedral angles of φ = –130° and ψ = 120°. A β-strand is a stretch of peptide consisting of amino acids with a repeating hydrophobic (H), hydrophilic (P) residue pattern (HPHPHP…) with the backbone in an almost fully extended conformation. β-Strands are arranged adjacent to other strands and form an extensive hydrogen bond network with their neighbors, in which the NH groups in the backbone of one strand form hydrogen bonds with the CO groups in the backbone of adjacent strands. Therefore, unlike α-helices that form intrahydrogen bonds, β-sheets form interhydrogen bonds. Because peptide chains are directional, with an N- and C-terminus, β-strands are also directional and can form sheets in a parallel, antiparallel, or mixed arrangement, which are shown in Figure 2(b). They form a pleated structure, with the side chains of the peptide sequence pointing alternately above and below the plane of the β-sheet. With the alternating HP pattern, one face of the β-sheet contains hydrophobic residues while the other contains hydrophilic residues. Analogous to the case of amphipathic α-helices, this allows for the formation of higher order structures.

7.08.2.3 Tertiary Structures

Proteins perform a diverse array of functions and must recognize many thousands of different molecules in the cell by detailed 3D interactions. These diverse, irregular, 3D protein structures that nature has evolved are termed tertiary structures, and are determined by the primary structure and the different possible ways to pack the regular secondary structural elements. It is rather impressive that nature has evolved to rely on the arrangement of the few common secondary structural elements to give rise to a large library of proteins that exist. The tertiary structure of a protein refers to the 3D structure of the folded protein, and it is usually the simplest structural element capable of performing a function. Secondary structural elements pack against each other to form simple tertiary structural motifs, and several motifs usually combine to form compact folded protein structures, also known as quaternary structures. There exist many different tertiary structures; two well-known examples, based on α-helices and β-sheets, are coiled-coils and β-barrels. They are commonly found in proteins and are key to such functions as enzymatic activity, transport, signaling, and redox reactions.

7.08.2.3.1 Coiled-coils

A common tertiary motif based on α-helices is the coiled-coil, as shown in Figure 3. Coiled-coils are left-handed supercoils of multiple right-handed α-helices. Each helix is amphipathic, with a hydrophobic and a hydrophilic face, and has a characteristic heptad repeat, labeled abcdefg, shown in Figure 3(a). The a and d positions of the helical wheel are usually occupied with hydrophobic residues so that the side chains from multiple peptides can pack together to form the hydrophobic core of the bundle, giving rise to much of the stability and specificity of coiled-coils. Residues in positions e and g border the hydrophobic core and are typically charged residues that form salt bridges between helices that determine the relative chain alignment and orientation. In the case of globular proteins, the rest of the residues are exposed to water on the exterior of the coiled-coil and are typically made up of hydrophilic amino acids. Therefore, the periodicity of hydrophobic and hydrophilic residues of a coiled-coil peptide follows the pattern, HPPHPPP.8,9

Figure 3. (a) Schematic drawing of the heptad (abcdefg) repeat of a coiled-coil 3-helix bundle. The a and d residues are hydrophobic and well packed in the interior of the helix bundle, colored green. (b) Crystal structure of a coiled-coil 3-helix bundle, known as 1coi in the Protein Databank.

7.08.2.3.2 β-Structures

A common tertiary motif composed of β-sheets is the β-barrel. A β-barrel is a large β-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last, as depicted in Figure 4. β-Strands are typically antiparallel, with hydrophobic residues oriented in the interior of the barrel to form a hydrophobic core and the polar residues oriented toward the exterior of the barrel. Membrane proteins containing β-barrels reverse this pattern, with hydrophobic residues oriented toward the exterior where they interact with surrounding lipids, and hydrophilic residues oriented toward the interior.

Figure 4. (A) Schematic drawing of a β-barrel. (B) Model of hierarchical self-assembly of β-sheets. (c–f) Local arrangements and (c′–f′) the corresponding global equilibrium conformations for the hierarchical self-assembling structures formed in solutions of (a) β-sheets, which align to form (c) tapes, then (d) twisted ribbons, then (e) fibrils, and (f) fibers. (e and f) The front views of the edges of fibrils and fibers, respectively.

β-Sheet structures are also commonly recognized and studied in their amyloid form. These amyloid structures are insoluble aggregates that are linked to a range of diseases, most notably Alzheimer’s disease.10–12 They form from the stacking of β-strands that lie perpendicular to the axis of the fibril. β-Strands are hydrogen bonded to form β-sheets that run parallel to the fibril axis. These stacks of β-sheets then bundle together to form the fibril. The diameter of most fibrils tends to be around 10 nm, with its length varying drastically. Because of the harmful effects linked to such amyloidosis, much research has been done in this area to gain fundamental understanding of the structure and the interactions governing their self-assembly, with the eventual goal of developing a cure for linked diseases. The increased knowledge of β-sheet structures has also led the materials scientist to use them as useful nanostructured materials. They are attractive because they form the distinct hierarchical levels of structure that span length scales from nanometers to micrometers, as β-strands assemble to form β-sheets, which further stack to ribbons, then fibrils, to fibers with a well-defined structure, shown in Figure 4.13 Though most of the work to date in the field of using peptides as materials has been based on such β-sheet structures, they will not be discussed further here because there already exist many comprehensive references.14,15 Rather, we will focus largely on helix-based structures because we believe they hold great promise and require further emphasis in the field.

7.08.2.4 Stability of Protein Folds

The conformational stability of a protein is mainly governed by the amino acid sequence in a given environment. Local conditions, such as solvent, pH, and ionic strength, can modulate the conformational properties of a protein. The role of solvent is of particular importance for protein structure stabilization, so globular proteins in aqueous media and membrane proteins embedded in lipid bilayers require different consideration. The hydrophobic effect, which is the tendency of water to exclude apolar molecules, resulting in segregation of water and apolar amino acids, is the major driving force in protein folding. Globular proteins are surrounded by water molecules, so the main driving force for folding is the packing of hydrophobic side chains into the interior of the molecule, thus creating a hydropohobic core and a hydrophilic surface. The stabilization of protein structures in water represents a delicate balance between the conformational entropy of the polypeptide chain, which favors unfolding, and various stabilizing energetic contributions, such as hydrogen bonds, and hydrophobic, van der Waals, and electrostatic interactions. The energy scales for each of these contributions are small, on the order of kT, so fluctuations in the environment, such as pH, ionic strength, and temperature changes, may have a large impact. In order to pack hydrophobic side chains in the interior of a protein, the backbone must also fold into the interior. The main chain is highly polar and therefore hydrophilic, so there is a great energetic penalty for removing it from the polar environment. This is alleviated by the formation of intrahydrogen bonds between the backbone NH and CO groups by the folding of secondary structures.

Membrane proteins, on the other hand, are embedded in the interior of a lipid bilayer, so the presence of hydrophilic residues on their exterior is energetically unfavorable. The hydrophobic interior of a lipid bilayer, like the hydrophobic core of a protein, provides no hydrogen bonding donors or acceptors. The energetic penalty for inserting a polar amino acid into the lipid bilayer can be very substantial, unless the hydrogen bonding capacity of the group is satisfied. Therefore, all membrane-spanning segments form secondary structures, with polar side chains rarely inserted into the lipid bilayer. The unique conformation of a native protein is determined by a large number and a large variety of noncovalent interactions, which severely restrict the possible conformational space that is available due to rotations around each single bond of the backbone. A thermodynamically stable conformation arises from the minimization of the overall free energy of interaction resulting from all the intra- and intermolecular contributions.

7.08.2.5 Peptides as Building Blocks for Hybrid Materials

Peptides are a desirable class of building blocks because they are able to self-assemble into the native structure that is encoded by their primary sequence. This well-defined structure leads to a diverse and complex array of functions. Therefore, the main advantages of peptides or proteins include hierarchical self-assembly, chemical functionality, selectivity and specificity, and dynamic response to external stimuli. The hierarchical structure provided by peptides allows for precise and well-defined building blocks that are identical from one to the next and are ordered over multiple length scales, from the molecular level to higher order tertiary structures. The chemical functionality possible with proteins is exemplified by the diverse functions that nature has evolved, as proteins are essential to organisms and participate in virtually every cellular process. For example, proteins are important for catalyzing enzymatic reactions, cell signaling, transport, cell adhesion, and the cell cycle. They also play major structural roles. For example, cytoskeletal proteins are responsible for assembling and disassembling a scaffold that maintains cell shape and controls cell motility. A majority of these functions relies on the protein’s ability to bind other molecules specifically and with high affinity. This binding capability is mediated by the tertiary structure of a protein, which defines the binding pocket. The structures and interactions of biomolecules are largely dictated by noncovalent interactions. They are designed to undergo dynamic and reversible conformational transformations upon changes in pH, temperature, and ionic strength. Each of these can be varied to tailor or control the structure of the protein, so that stimuli responsiveness can be engineered, possibly leading to responsive materials. Because all of these characteristics of proteins are largely determined by structure, it is important to retain the natural structure of a protein, and thus its functionality, when attempting to incorporate it into hybrid materials.

In the case of synthetic polymers, they are usually composed of only a few types of monomers, which limits their chemical heterogeneity. Although polymers can form hierarchical structures, it is difficult to obtain atomic level structural control and built-in functionality at the molecular level. Figure 5 depicts the difference in hierarchical assembly between peptides and synthetic block copolymers (BCPs). While BCPs can form micellar aggregates with a simple core-shell fine structure, peptide organization leads to distinct nanostructures with precise hierarchical inner structures, from primary to quaternary structures. There exist many possibilities available for the biomolecular block, as exemplified by the various protein structures found in nature. To provide a concise survey of the field, the remainder of this chapter will focus largely on the use of helix-based peptides as building blocks for hybrid materials, with an emphasis on coiled-coil helix bundles as a promising option.

Figure 5. Schematic illustration of the structure formation processes in (a) polypeptides and (b) amphiphilic AB block copolymers. While the block copolymer forms micellar aggregates with a simple core-shell fine structure, the peptide organization leads to distinct nanostructures with a precise hierarchical inner structure (‘primary structure’: the amino acid sequence of the peptide chains; ‘secondary structure’: locally defined substructures in a single protein molecule; ‘tertiary structure’: spatial arrangement of the secondary structures in a 3D structure of a single protein molecule; and ‘quaternary structure’: arrangement of tertiary structure subunit assembly).

7.08.2.6 The Promise of Coiled-Coils

Engineering minimalist protein structures that self-assemble and reproduce native-like function, thus simplifying the system from a complex, globular protein to a relatively short, precisely folded peptide is a valuable strategy for incorporating biomolecular building blocks to generate useful materials.16 The coiled-coil is one of the simplest tertiary structural motifs and is the foundation of many of the functionalities of natural proteins, such as enzymatic activity, signal transfer, and redox chemistry.17,18 Relatively short peptides can self-assemble into a coiled-coil to form a simplified version of a globular protein that is more robust and better-defined in structure, while still being able to perform the specific function of the native protein.

Peptide–polymer conjugates based on coiled-coil peptides present a versatile strategy for generating hybrid functional materials.19–21 BCPs containing leucine zippers, a common coiled-coil, have been used to prepare hydrogels where the formed helix bundle acts as a physical cross-link.19–21 Coiled-coils have also been used as carriers for drug and gene delivery, as shown in Figure 6(a).22,23 As a structural motif, though, the functionality of coiled-coils goes far beyond what has been explored to date, so there exists a lucrative area for further study. For example, they should be able to perform highly selective transport and enzymatic reactions and transduce signals to activate downstream reactions.24–26 The selectivity, sensitivity, and responsiveness afforded by coiled-coils are far superior to those possible with synthetic materials alone, and should thus be exploited.

Figure 6. Examples of de novo designed coiled-coils and their applications. (a) Schematic illustration of the structure of a proposed noncovalent poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA)-based polymer therapeutics and its envisioned pathway for cell uptake and subsequent intracellular release of the cargo.

Reprinted with permission from Apostolovic, B.; Deacon, S. P. E.; Duncan, R.; Klok, H. A. Biomacromolecules 2010, 11, 1187–1195.23 Copyright © 2010, American Chemical Society. (b) Schematic drawing of a de novo designed coiled-coil 4-helix bundle capable of binding a conjugated porphyrin. Reproduced with permission from Ye, S. X.; Discher, B. M.; Strzalka, J.; et al. Nano Lett. 2005, 5, 1658–1667.31 Copyright © 2010, American Chemical Society. (c) Schematic drawing of a de novo designed coiled-coil 4-helix bundle that is designed to selectively bind a nonbiological cofactor. Upon binding the nonbiological cofactor, the peptide goes through a random coil-to-helix transition.Reproduced with permission from Cochran, F. V.; Wu, S. P.; Wang, W.; et al. J. Am. Chem. Soc. 2005, 127, 1346–1347.35 Copyright © 2010, American Chemical Society.

De novo protein design (design with novel amino acid sequences) also provides tremendous versatility and tailorability in the use of coiled-coils as building blocks.8,17,27,28 Helix bundles derived from natural proteins can be tailored to retain specific functionalities,29–31 while novel peptide sequences can be de novo designed to mimic natural proteins or to obtain properties not seen in nature.32–37 These peptides have both chemical and structural diversities that are comparable to or greater than those found in natural materials, thereby significantly expanding the repertoire of these building blocks by enabling their design to meet specific needs. In addition, the interior of the coiled-coil, which is key to its functionality, can be tailored independently of the exterior of the coiled-coil, which governs its interaction with the environment, as shown in Figure 3. Therefore, a sequence may be designed so that a peptide with a desired structure and functionality can be incorporated into the environment of choice.27 For example, the amino acids on the exterior of the transmembrane region of a natural protein have been mutated to modify the protein from being lipid to water soluble without interfering with its built-in functionality.38 To mimic the function of natural proteins, such as signaling or enzymatic reactions, the bundle interior can be designed to bind specific prosthetic groups with high selectivity and precision in their spatial arrangement, and the dissociation constants can be tailored from the subnanomolar to the micromolar, as depicted in Figure 6(b).31,34,36,37,39–41 A large library of coiled-coils is readily available, capable of transporting protons or positioning chromophores for light-induced charge separation.32–34,42,43 Helix bundles can also be de novo designed to incorporate stimuli responsiveness. Figure 6(c) shows a coiled-coil 4-helix bundle that was designed to undergo a random coil-to-helix transition when it selectively binds a nonbiological cofactor.35 These recent developments in de novo protein design clearly demonstrate the possibility to custom design building blocks with novel properties that are not possible with synthetic materials alone.

De novo designed peptides, in general, are much more robust in comparison to their natural counterparts. Folding for a short peptide sequence is a much simpler process compared to that for large natural proteins.44 For most de novo designed peptides, folding is fully reversible,45 and they tend to have better stability and can maintain their structure against changes in temperature and solvents.46 Thus, they can retain their designer functionalities under conditions that are unsuitable for their natural counterparts.34 However, they are still subject to proteolysis and degradation, and it remains a challenge to process them into functional materials over macroscopic distances.46,47 To take full advantage of the unique properties offered by coiled-coils, controlling their macroscopic assembly is essential. For example, helix bundles must be oriented normal to a membrane or substrate interface in order to mimic natural transmembrane proteins. Various strategies include designing distinctive charge patterns and hydrophilic/hydrophobic patterns along the exterior of a coiled-coil so that they can be readily inserted into vesicles or macroscopically oriented at polar/nonpolar interfaces.48–51 However, studies of a Langmuir monolayer at the air/water interface showed that the packing of amphiphilic helix bundles is only liquid-like, showing no 2D ordering.47

Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B9780444533494001898

1 Introduction

During the last few decades, the number of peptide and protein drugs have increased dramatically primarily due to advances in recombinant DNA technology and hybridoma techniques. Small peptides are increasingly used in the prevention and treatment of numerous diseases such as chronic pain, hypertension, memory loss, cancer, epilepsy, diabetes, and other disorders [1]. Importantly, peptides act as intrinsic signaling agents for various physiological functions and present an opportunity for therapeutic intervention. Generally, peptides are efficacious signaling agents that bind to specific receptors, such as ions channels or G protein-coupled receptors (GPCRs), where they initiate intracellular effects. Thousands of naturally occurring peptides have been reported and many of these have significant role in human physiology [2].

Cryptome is a subset of the proteome which constitutes cryptic peptides with unique biological functions [3]. When subjected to proteolytic cleavage, the cryptome, typically embedded in protein sequences, has the ability to produce several bioactive peptides called cryptides [3,4]. Cryptides could have similar or, often, completely different biological functions when compared to the protein from which it originated. Recently, several cryptides have been identified and their putative biological functions in numerous disorders have been reported [5–10]. Hemorphins are endogenous cryptides, belonging to the family of atypical opioid peptides, released during the sequential cleavage of hemoglobin proteins [11,12]. Natural opioid peptides are broadly classified into typical and atypical opioid peptides. Enkephalins, beta-endorphins and dynorphins belong to the classical or typical opioid peptide group. These peptides are produced by the cleavage of proenkephalin, proopiomelanocortin and prodynorphin, respectively, and have the same N-terminal amino acid sequence tyrosine-glycine-glycine-phenylalanine [13–16]. Controlled lysis of certain proteins results in the release of another group of peptides called as atypical opioid peptides. Hemorphins, cytochrophins, and casomorphins derived from hemoglobin, mitochondrial cytochrome b, and casein, respectively, belong to this group [17–19]. The only common factor is an N-terminal tyrosine residue. In contrast to typical opioid peptides, further extension of the amino acid sequence beyond the Tyr residue at the N-terminal is possible in atypical opioid peptides [20]

Hemorphins are between 4–10 amino acids long and are produced by the cleavage of hemoglobin proteins [17]. Hemorphin-4 was the first characterized hemoglobin-derived peptide isolated from bovine blood [18]. Subsequently, Glamsta and colleagues reported extended forms of naturally occurring bioactive hemorphins in the human pituitary [21]. Sequential degradation of hemoglobin produces a chain of bioactive peptides with overlapping sequences. These bioactive peptides have been identified and isolated from various biological fluids such as plasma, cerebrospinal fluids, brain, hypothalamic tissues, peripheral and central nervous systems, pituitary gland, and adrenal tissue [22–25]. These peptides have been shown to be associated with physiological and pathological conditions such as long distance running, inflammation, cerebrovascular bleeding, and cancer [22,26–29]. Reports have demonstrated that hemorphins are produced during alterations from normal behavior and the activity of certain enzymes in specific tissues [19,30]. These short peptides share a central tetrapeptide core - tyrosine-proline-tryptophan-threonine (YPWT) [31]. Peptides with N- and C- terminal extensions of this core sequence have been isolated from human and bovine tissue [31]. A list of known hemorphin peptides is given in Table 1. Among all reported hemorphins, LVV-hemorphin-7 and VV-hemorphin-7 are considered the longest and most stable forms of hemorphins [32,33]. Being relatively small, hemorphins have also been observed to pass across the blood-brain barrier (BBB) [22]. A large body of evidence suggests that hemorphins could have a therapeutic role in several disorders [34–38]. However, the precise cellular and molecular mechanisms that underlie the interaction of hemorphins and their targets remain largely unknown.

Table 1. Sequence of hemorphin peptides.

Name	Amino acid sequence
Hemorphin-4	Tyr-Pro-Trp-Thr (YPWT)
Hemorphin-5	Tyr-Pro-Trp-Thr-Gln (YPWTQ)
Hemorphin-6	Tyr-Pro-Trp-Thr-Gln-Arg (YPWTQR)
Hemorphin-7	Tyr-Pro-Trp-Thr-Gln-Arg-Phe (YPWTQRF)
LVV-hemorphin-4	Leu-Val-Val-Tyr-Pro-Trp-Thr (LVVYPWT)
LVV-hemorphin-5	Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln (LVVYPWTQ)
LVV-hemorphin-6	Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg (LVVYPWTQR)
LVV-hemorphin-7	Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (LVVYPWTQRF)

This review focusses on key studies that have bettered our understanding of how hemorphins behave at the cellular and molecular level to bring about its beneficial effects. Here, we consolidate the molecular basis of hemorphins in several pathological conditions and suggest possible avenues for future work. Emphasis is given to the effects of hemorphins on the renin-angiotensin system (RAS), opioid receptors and insulin-regulated aminopeptidase receptor (IRAP).

Read full article

URL: https://www.sciencedirect.com/science/article/pii/S1043661820311634

5.4 Polyethylene Glycol-Peptide Conjugation

Peptides are fundamental components of cells that provide important biological functions and regulate major cell activities. Peptides consist of 2–50 amino acid chains linked by amide bonds known as peptide bonds, and are stabilized by disulfide bonds with other molecules [101]. Peptides are named according to the presence of amino acids (e.g., dipeptide, tripeptide, tetrapeptide, oligopeptide, and polypeptide). Synthetic peptides are synthesized artificially according to requirements and natural peptides can be obtained from proteolysis of the proteins in the body. There are more than 7000 natural peptides already discovered that have crucial roles in human physiology [102]. Due to their excellent pharmacological properties, tolerability, and specific targetability, they are attractive for therapeutic treatment of diseases. Therapeutic peptides have greater advantages than proteins and antibodies. They are considered preferable agents because of their smaller size, easy synthesis, excellent penetration ability, greater specificity, and variety of biological and chemical activities [102]. Peptides are easily modified according to need, and they are not accumulated in specific organs like kidney and liver, which indicates their less toxic character [103]. Using therapeutic peptides to treat various types of cancers is a novel and favorable idea for making an effective anticancer drug. Peptides are used as drug carriers for anticancer drugs, hormones, and radionucleotides for chemoradiation therapy. Their ability to bind to different receptors on the cell surface makes peptides novel agents for targeting therapy and organized biosensors for cancer therapy [101]. The therapeutic activity of the peptides used for cancer treatment is divided into three groups: antimicrobial pore-forming peptides, cell-permeable peptides, and tumor-targeting peptides. Antimicrobial pore-forming peptides are natural peptides that have roles in innate immunity and that target cancer cell membranes to induce apoptosis and necrosis. Cell-permeable peptides with length of 2–30 amino acids can penetrate a cell through the plasma membrane to transport small cellular components. Tumor-targeting peptides can target many receptors that are overexpressed in tumor cells, and thus are promising as biomarkers for targeted therapies [104].

There are some limitations for peptide-based cancer therapy that include rapid elimination, chemical instability, tendency to aggregation, and high immunogenicity that obstructs treatments [105]. Many therapeutic studies have already addressed ways to overcome the problems regarding this therapy. PEGylation is a potent method for increasing the in vivo half-life and reducing the immunogenicity of the therapeutic peptides used for treatments. There are many synthetic methods for coupling a PEG moiety (shown in Table 10.1) to the peptides to stabilize the peptide activity without lowering its effect. Amino or hydroxy groups of functionalized PEGs are suitable for coupling of the peptide through the N terminus or lysine side chains. Click chemistry also plays an important role in peptide coupling through alkyne and azide reactions [111]. Locating the site for PEG attachment to a peptide and the number of chains to be used should be carefully engineered to retain the highest binding affinity and treatment effectiveness. Conjugating PEG chains increases the molecular mass and solubility of the peptides [111]. This offers protection from proteolytic enzymes and improves the pharmacokinetics of the peptide by reducing inappropriate aggregation. PEGylation reduces dosing frequency of the peptide drugs, which can provide improved cost and therapeutic effect. Conjugation of linear, branched, or more complex PEG chains can be selected according to the appropriate application. Heterobifunctional PEG has different functional groups that can bind two peptide chains to improve cell binding and targeting affinity with higher pharmacokinetics properties. There is a list of peptides conjugated with PEG for various cancer treatment applications, as well as notes on their clinical status, in Table 10.1.

Table 10.1. List of PEGylated Peptides Currently Being Used for Cancer Therapy.

Peptide conjugate	Sequence	Cancer treatment	Clinical status	Functions	Reference
PEG-RGD	GCGYGRGDSPG	Brain, ovarian, prostate, melanoma	Phase I & II	Integrin targeting, blocking the new blood vessels in tumor	[106]
PEG-NGR	CNGRCG	Ovarian, sarcoma, lung, colon, hepatic	Phase II & III	Cd 13 receptor targeting	[107]
PEG-TAT	GRKKRRQRRRPQ	Breast	Preclinical	Cell penetration Delivery of proteins, DNA, antibodies, drugs inside cell	[108]
PEG-BH 3	LWAAQRYGREL-RRMSDEFEGSFKGL	Leukemia, cervical, prostate, glioma, lung, breast	Preclinical	Bcl2 binding peptide, inducing cell apoptosis	[109]
PEG- LyP1	CGNKRTRGC	Breast carcinoma, lung, metastatic, prostate	Preclinical	Mitochondrial p32 targeting	[110]
PEG-KLAKLAK	KLAKLAKKLAKLAK-NH2	Breast, metastatic	Preclinical	Mitochondrial membrane distortion	[111]

Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B9780128169636000108

Physicochemical Nature of Peptides

Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B0122267702037819

3 Design of peptide-based probes for PET imaging

A desirable PET imaging probe with clinical translation potential is expected to have the following unique characteristics: (i) high binding affinity to target; (ii) high specificity to target; (iii) high contrast ratio; (iv) good stability in vivo; (v) low immunogenicity and toxicity; and (vi) easy production [14]. A peptide is any combination of amino acids linked by peptide bonds. Generally, peptides considered as imaging agents have a low molecular weight, containing several to fewer than 50 amino acids. These small peptides have a number of distinct advantages over macromolecules, such as proteins and antibodies. Small peptides have favorable pharmacokinetic and tissue distribution patterns, as characterized by rapid clearance from blood and nontarget tissues. Some peptides have good permeability properties that can permit rapid access to target tissues. In addition, peptides usually have low toxicity and immunogenicity, and they are quite flexible in chemical modification and radiolabeling. A large variety of biologically active peptides radiolabeled with clinically useful PET radionuclides for diagnosis and therapy of various diseases have opened a new era in the field of nuclear medicine [26–28].

Development of peptide-based PET probes has customarily relied on (i) isolation of naturally occurring peptides, (ii) screening synthetic peptide libraries, and (iii) structure-based rational design. In general, most peptide-based PET probes are designed on the basis of naturally occurring peptides which play a vital role in physiological conditions mediated through their specific and high-affinity receptors. Many of these peptide-binding receptors are massively overexpressed in various diseases. However, naturally occurring peptides typically have a short biological half-life, due to rapid degradation by various peptidases and proteases found in plasma and in most tissues. Thus, in vivo stability is a key factor in designing peptide-based PET probes. After the indispensable amino acid residues that are involved in the biological activity have been determined, most naturally occurring peptides are molecularly engineered to prolong their biological half-lives in vivo. The approaches commonly used to improve the metabolic stability of peptides include the introduction of appropriate d-amino acids, substitution of peptide bonds, utilization of unusual amino acids or side-chains, incorporation of amino alcohol, and acetylation and/or amination of peptide N- and C-terminus. In addition, the rate of excretion and permeability of peptides can be modified by the introduction of specific hydro- and/or lipophilic amino acids or other chemicals into the peptide-chain without altering the binding properties. Lipophilic peptides are usually cleared from the body by the hepatobiliary route, whereas hydrophilic peptides are mainly cleared via the renal system. Furthermore, peptide cyclization can be applied to restrict the three-dimensional conformation of the peptide and to improve receptor binding affinity and stability. It must be emphasized that the biological functions of peptides are largely determined by their three-dimensional conformation. Modification of a naturally occurring peptide may cause significant loss of its biological activity. To retain the biological activity of peptides, a linker can be incorporated between the peptide and the radiolabeling moiety. Therefore, the side interaction of the radiolabeling moiety with the active binding site of peptide can be minimized. More importantly, the incorporation of an appropriate linker may have a profound impact on the biodistribution of the imaging probe [1,4,29–31]. To date, a large variety of linkers are being used to modulate the pharmacokinetics of the imaging probe. The length, flexibility, hydrophilicity, and charges (i.e., cationic, anionic, and neutral) are the key factors to be considered to select an appropriate linker.

An alternative technique for target-specific peptide discovery involves the screening of bacteriophage (phage) display libraries. Phage display is a powerful technique that allows vast sequence space screening, providing a means to improve peptide affinity and generate unique peptides that bind any given target [32]. Since its inception almost 30 years ago, phage display has been utilized in vitro, in situ, and in vivo. Detailed approaches for the discovery of targeting peptides by phage display technique have been summarized [32–36]. In brief, phage display libraries usually contain a vast array of different clones (approximately 107–109) that can be rapidly screened in an effort to select a target-specific peptide. The specific peptide will be amplified, characterized, and sequenced. The obtained target-specific peptide can be then radiolabeled with positron emitters for PET imaging [37,38].

Read full article

URL: https://www.sciencedirect.com/science/article/pii/S0169409X10001821

3 Properties of peptides

3.1 Structure of peptides and proteins

Amino acids are the building blocks of peptides and proteins. All AAs have the same basic structure. There are 20 different, naturally occurring amino acids. The properties of each amino acid are determined by its specific side chain. Accordingly, amino acids are categorized as polar, nonpolar, acidic and basic molecules. Amino acid names are often abbreviated as either three letters or single letters. See Table 1.

Table 1. Code letters, Hydropathy index and hydrophobic/hydrophilic properties of amino acids [60].

Peptides are composed of 50 or less amino acids(5000 Da MW), specified by a high level of secondary structure and lack of tertiary structure [61]. Some peptides are hydrophobic due to their contents of various hydrophobic amino acids. This hydrophobic characteristic helps peptides to show an effective biological activity against cancer tumors [59]. Peptides, polypeptides and proteins consist of 1–100, 100-20 and over 200 amino acids, respectively [62]. Oligopeptides with 2–20 amino acids (in some sources 2–50) are bioactive peptides that play important roles in body safety and immune system activity [63]. Table 2 shows the molecular weights of peptides in comparison with other molecules.

Table 2. Left to right shows size and molecular weight (MW) increasing from small molecule to biology cell [64].

Amino acids are joined together to make long polypeptide chains folded in spatial different structures [65]. Chains contain less than 40–50 amino acids are often referred to as polypeptides with less complexed structures.

3.1.1 Primary structure

The primary structure of a polypeptide or protein is characterized by the sequence of amino acids connected together to form a long chain. The two ends of each polypeptide chain are known as the amino terminus (N-terminus) and the carboxyl terminus (C-terminus). Twenty different amino acids can be used multiple times in a polypeptide to create a specific primary structure [66].

3.1.2 Secondary structure

The term secondary structure refers to the interaction of residues through hydrogen bonding within peptide chains. This structure is formed and directed by the wrapping (coiling) of the peptide long chain [67]. Two main types of secondary structures, α-helix and the β-pleated strand(β-sheets) are important in the activity of anticancer peptides [68]. The recognition of classical structures of proteins as α-helix, β-sheet, and random coil led to discovering important sections and concepts like domain and folds; but a new framework, termed turn structure developed thereafter, expanded these concepts [69].

3.1.2.1 Helix: helix is one of the most important secondary structures of peptide or protein which appears in different types3.1.2.1.1 α-Helix

α-helix involves a peptide chain coiled into a clockwise spiral conformation and fixed by hydrogen bonds between the CO group of each amino acid and the NH group of the other amino acid positioned four residues ahead in the sequence (Fig. 2) [70]. Alanine, leucine, glutamine, and lysine have special propensity to be part of α-helix structures while proline and glycine show weak helix-forming tendency [71]. Each of the 20 amino acids has a specific side chain, known as an R group and attached to the α carbon next to the carboxylate group (Fig. 2). The R groups are different in shapes, sizes, charges, and reactivity [72].

Fig. 2. Structure of α-Helix from far and near angles. α-Helices are regular right-hand turns of 3.6 residues long, 5.41 Å. Hydrogen bonding between the first backbone carbonyl oxygen atom and the fourth residue NH group stabilizes the structure; The van der Waals forces along the axis make the structure more stable [73].

3.1.2.1.2 310-Helix

In this type of helix, N–H group of an amino acid forms a hydrogen bond with the CO group of the other amino acid positioned in three residues before/after [74]. These structures are much less probable and less stable than general α-helices; they are not normally favored [75]. 310-Helix is the fourth most common type of secondary structure in proteins after α-helices, β-sheets, and reverse turns [76]. This type of helix probably mediates between folding and unfolding of α-helices [77].

3.1.2.1.3 Pi-helix(π-helix)

In this type of helix, N–H group of an amino acid forms a hydrogen bond with the CO group of an amino acid resided in five residues farther [78]. The pi-helix plays a significant role in translating structure into function [79].

3.1.2.2 Beta sheets

Beta sheets are a more spacious type of secondary structure formed by β-strands. The strands include 4 to 10 amino acids. Single β-strands are not energetically favorable. However, they can form β-sheets which are more stable characterized by a pattern of hydrogen bonding between residues located in the two different β-strands, parallel and antiparallel [80]. In beta sheets, the backbones’ groups N–H and CO establish hydrogen bond with the CO and N–H groups of lateral strand, respectively [81]. Alpha helix is a self-sustaining secondary structure that involves a significant number of amino acids, while beta-strands generally involve 3 to 6 amino acids and require an adjacent strand to form a stable folded structure [82].

3.1.2.2.1 Parallel

Parallel β-pleated sheets, in which the polypeptide chains run in the same direction, have less-stable hydrogen bond than anti-parallel β-sheet.

3.1.2.2.2 Antiparallel

Antiparallel β-sheet structure is an essential structural part of native protein. Most sheets in proteins’ structures tend to be antiparallel [75]. Antiparallel β-sheet exists in many native proteins such as silk [81]. β-Hairpins are secondary structures formed by two antiparallel β-sheets [83]. Parallel sheets are less twisted than antiparallel ones. On the other hand, antiparallel sheets can withstand greater distortions (twisting and beta-bulges) and greater exposure to solvent than parallel sheets can.

3.1.2.3 Turns

β-Turn is the third most important secondary structure following helices and β-strands [84]. Among the various types of turn, β-turn is the most prevalence type [85]. Actually, turns interconnect secondary structural elements (Fig. 3).

Fig. 3. β-turn, secondary structure of peptides.

3.1.2.4 Random coils

This category of the peptide structures involves miscellaneous shapes and, in fact, any structure that is not considered within the above classes can be considered in this category.

Since alpha helix is the most important spatial structure in anti-cancer peptides, it is important to consider the tendency of different amino acids to help formation of this structure. Table 3 shows comparative tendency of amino acids to form helix.

Table 3. Amino acids tendency for alpha helical formation [86].

No	Amino acid	Helical penalty Kcal/mol
1	Alanine	0.00
2	Arginine	0.21
3	Asparagine	0.65
4	Aspartic acid	0.69
5	Cysteine	0.68
6	Glutamic acid	0.4
7	Glutamine	0.39
8	Glycine	1.00
9	Histidine	0.61
10	Isoleucine	0.41
11	Leucine	0.21
12	Lysine	0.26
13	Methionine	0.24
14	Phenylalanine	0.54
15	Proline	3.16
16	Serine	0.50
17	Threonine	0.66
18	Tryptophan	0.49
19	Tyrosine	0.53
20	Valine	0.61

Aromatic AA such as tyrosine, phenylalanine, tryptophan and β-branched amino acids such as threonine, valine, isoleucine are found in the middle of β-sheets and some other AAs like proline are found in the edge strands of β-sheets, probably to prevent the “edge-to-edge” association between proteins molecules that might lead to aggregation and amyloid formation [87–89]. Proline is distinctive in its cyclic structure among amino acids encoded from ribosomes and commonly found in the turn structure of proteins [90]. Proline forms tertiary amide when combined with biopolymers, thus inhibit hydrogen bond formation. Despite conformational restriction and the lack of hydrogen bonding due to the pyrrolidine ring, this amino acid is able to handle peptide secondary structures such as β-turns or polyproline helices [91].

3.1.3 Tertiary structure

In proteins, the secondary structure folds to form a three-dimensional structure known as the tertiary structure, or commonly three-dimensional conformation of the proteins. There are many chemical bonds which maintain and stabilize the tertiary structures of proteins including: ionic bonds (between NH3+ and COO-), hydrogen bonds, hydrophobic/hydrophilic interactions and disulfide bridges [92]. The third structure is obtained from the folding of the second structure, and called the domain. Based on this structure, proteins can be divided into two categories: fibrous and globular, the first one involves elongated structures which come together to form filament or bundle shape with hydrophobic characteristic, while the second one engages global forms which are hydrophilic [93]. The tertiary structure of proteins shows how the regional structures gather in space. Accordingly, the α-helices may be oriented parallel or perpendicular to each other. Hence, the tertiary structure indicates folding of the variety of protein sectors as helices, sheets, turns, and other parts which come together into the natural three-dimensional structure [94].

3.1.4 Quaternary structure

If two or more tertiary structures of proteins form a single block, this refers to the quaternary structure. This structure, in fact, involves subunits in a nearly packed form. Each of the subunits contains its’ own primary, secondary, and tertiary structure. The subunits are interconnected by hydrogen bonds and van der Waals forces. Insulin and hemoglobin are those proteins that have quaternary structures [95].

3.2 Chemical bonds that play vital roles in 3D structure of peptide/protein

3.2.1 Covalent bond is the equal sharing of an electron pair by two atoms [96]

Covalent bonds are the strongest bonds in nature and under normal biological conditions may only be broken with lytic enzymes [97].

3.2.1.1 Peptide (amide) bonds are important forms of covalent bond between amino acids which include C–C, C–O, and C–N bonds3.2.1.2 Disulfide bonds [-S-S-] are formed between amino acids which contain thiol groups

Cysteine which contains a thiol group (-SH), plays a great role in biological system [98]. The protons of the both SH groups are lost very easily in an oxidation reaction and remaining sulfur groups join together making a disulfide bond. Thiol (sulfhydryl) groups are also involved in complexation with metal cations present in the active site of many enzymes, and play a crucial role in enhancing the catalytic activity of enzymes. Although sulfhydryl groups typically have a neutral charge, they are polar and hydrophilic, thus reacting in aqueous solutions [99]. Disulfide bonds are made in nearly one-third of the proteins in the eukaryotic proteomes [100].

3.2.2 Coordinate covalent bond

This bond involves the unequal sharing of an electron pair between two atoms, with an electron pair donating from one atom towards an electron acceptor atom. The electron donor maybe a ligand and the electron acceptor as a core (central) element in a complex structure of macromolecules (because this element can repeatedly accept more than one pair of atoms). These bonds are essential in all interactions between transition metals and organic ligands (e.g., Fe2+ in hemoglobin and cytochromes) [101].

3.2.3 Noncovalent bond

Noncovalent bonds generally make weak interactions, but they can add up together to produce a raised acceptable stability of the molecules [102]. The three fundamental noncovalent bonds are electrostatic, hydrogen, and van der Waals bonds. They differ in geometry, strength, and specificity. Furthermore, these bonds are greatly affected by various ways in the presence of water [70].

3.2.3.1 Ionic bonds (salt bridges)

In ionic bonds, the complete transfer of electrons occurs from the donor atoms towards acceptor atoms [103]. Ionic bonds occur between losing electron metals and gaining electron nonmetals. Ions, thus formed with opposite charges, will attract each other making an ionic bond. Such bonds are stronger than hydrogen bonds, but weaker than covalent bonds [104]. Salt bridge is a product of the combination of two opposite ions. The salt bridge most often forms between the anionic carboxylate (RCOO−) group of either aspartic acid or glutamic acid and the cationic ammonium (RNH3+) and the guanidinium (RNHC(NH2)2+) groups of lysine and arginine, respectively [102]. This bond occurs constantly in proteins where cooperating in specificity, identification, and catalytic processes of them [105].

3.2.3.2 Hydrogen bonds

Hydrogen bonds are a type of dipole-dipole interaction formed between any X group-attached proton, where X is an electronegative atom, and an electronegative atom (Y) containing a pair of nonbonded electrons. The important hydrogen bonds occur in molecules where X and Y are N, O, or F atoms [106]. In proteins, groups possessing a hydrogen atom that can be shared from include a –NH (peptide nitrogen, imidazole, and indole),-SH (cysteine), –OH (serine, threonine, tyrosine, and hydroxyproline), –NH2 and –NH3+ (arginine, lysine, and N-terminal amino), and –CONH2 (carbamino, asparagine, and glutamine) groups. Those groups capable of accepting the sharing hydrogen atom include –COO–(aspartate, glutamate, and C-terminal carboxylate), -S-CH3(methionine), -S-S-(disulfide), and –CO (in peptide and ester linkages) groups [101].

3.2.3.3 Hydrophobic interactions

Hydrophobic bonds are a major force driving proper protein folding (Fig. 4). Hydrophobic interactions are responsible for relations between water and hydrophobic molecules (low water-soluble molecules). Hydrophobic molecules are nonpolar molecules and usually have a long chain of carbons that do not interact with water molecules. Fat in water medium is an example. Nonpolar substances like fat molecules tend to clump up together rather than distributing themselves in a water medium, because this allow the fat molecules to have minimal contact with water. Hydrophobic interactions are relatively stronger than other weak intermolecular forces (i.e., Van der Waals interactions or hydrogen bonds) [107–110].

Fig. 4. Hydrophobic Interactions within peptide chain forced by a heterogeneous medium (i.e., water) [111].

3.2.3.4 Van der waals forces

The van der Waals interactions occur when neighboring atoms or molecules come near together enough that their outer electron shells nearly contact [112]. When polar and nonpolar atoms or molecules are far from each other, van der Waals interaction is attractive. In contrary, when atoms or molecules are near to each other, van der Waals interaction is repulsive due to the repulsion effect of electron shells of atoms or molecules. Electrostatic repulsion occurs between groups with the same charge [65]. There are three kinds of van der Waals interaction including:

3.2.3.4.1 London dispersion force (induced dipole-induced dipole interaction)

London forces are responsible for attractive interactions between nonpolar molecules. The occurrence of these short-range interactions is due to the fact that any atom will, at any given instant, be likely to possess a finite dipole moment as a result of the movement of electrons around the nuclei. When molecules are approaching each other, the temporary dipoles of one molecule induce opposite dipoles in the other approaching molecules, thus resulting in a net attractive force [113].

3.2.3.4.2 Keesom force (dipole-dipole interaction)

Keesom forces are electrostatic interactions between two permanent dipoles that arise between two polar molecules which mutually modify their orientations to align their dipoles [114]. They arise when the δ+ end of a dipole in one molecule is attracted to the δ-end of a dipole in another molecule. This interaction is stronger than the London forces [115].

3.2.3.4.3 Debye force (dipole-induced dipole interaction)

Debye interactions are defined as the attractive force between a polar molecule and a nonpolar molecule [114]. When a permanent dipole molecule induces polarity on nearby nonpolar molecule, the kind of interaction between these molecules is called Debye force. Unlike Keesom interactions, Debye forces are not dependent on temperature [115]. Fig. 5 shows various chemical bonds in peptide or protein structure.

Fig. 5. Types of chemical bonds in protein structure.

3.3 Peptide entry into cell

Two striking entry mechanisms are, indirect penetration (endocytosis(macropinocytosis, clathrin or caveolin-mediated endocytosis, and clathrin/caveolin-independent endocytosis with enhanced endosomal escape from a lysosome)) and direct penetration causing membrane destabilization which includes different mechanisms such as inverse micelle, carpet model, membrane-thinning model, pore formation and so on [19,116]. The most frequently used CPPs include Tat, oligoarginine, and antennapedia (Antp) [117]. CPPs are also employed as drug delivery vehicles because of their excellent ability to facilitate cell uptake and overcome multidrug resistance (MDR) although they exhibit less specificity than receptor-targeting peptides [118]. As drug delivery vehicles, both receptor-targeted peptides and CPPs have different mechanisms for drug delivery to the tumor site. The hydrophobic nature of the cell wall makes a barrier for cell entry. However, receptor-targeted peptides can enter to the cell membrane by receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis using active transport. Therefore, anticancer substances can be conjugated to the peptides that act as ligands and bind to their cognate receptors and activate the energy-dependent endosomes in the cell. The endosomes are then taken inside the cell by lysosomes. It is followed by drug releasing into the cytoplasm.

3.3.1 Direct penetration

It is an energy-independent process and may include inverted micelle formation, pore formation, the carpet-like and membrane thinning model. The first step is interaction between peptide (with positive charge) and the membrane components (with negative charge) including heparan sulfate (HS) and phospholipid bilayer. The next step mechanism of internalization correlates mainly with the peptide backbone and lipid content of membrane under study [119].

3.3.1.1 Inverted micelle

Inverted micelles are formed to reduce the energy potential of the peptide. For vesicle membranes, peptide enters vesicle from the outside to the inside. This translocation is considered as reversed micelles, which is one of the possible mechanisms of anti-cancer peptides’ action [120].

3.3.1.2 Pore formation

Pore-forming peptides are of interest for providing a way to enter the lipid membrane. These peptides can be introduced by employing different strategies [121].

3.3.2 Indirect penetration3.3.2.1 Endocytosis

In endocytosis, the plasma membrane wraps around the extracellular material and folds to form a sac as a internalized vesicle [122]. Endocytosis generally includes pinocytosis (cell drinking) and phagocytosis (cell eating). It is a form of active transport (energy dependent). Endocytosis is the process used to transfer extracellular material by the cell membrane into the cell. There are various types of endocytosis involved in internalizing different types of cargo from hormone and nutrient receptors to large extracellular material such as bacteria [123].

3.3.2.1.1 Phagocytosis

Phagocytosis (literally, “cell eating”) is a form of endocytosis [124]. Phagocytosis is function of a cell, which starts with the recognition of a particle (over 0.5 μm in diameter), binding to, typically in a receptor-dependent state, and ultimately internalizing and degrading it [125]. Phagocytosis mainly occurs in specialized cells, such as immune cells, that need to internalize big particles, cell debris, or even unicellular organisms. Macro pinocytosis mainly involve the uptake of nutrients and fluids [126].

3.3.2.1.2 Pinocytosis

Pinocytosis, a non-specific, unsaturated method, with no intervention of a carrier, is the vesicular adsorption of fluid and its transportation to the cell from the surrounding medium [127], In other words, pinocytosis is a type of endocytosis that includes a fluid containing soluble substances. In humans, this process occurs in the cell lining of organs’ wall such as small intestine and is primarily used to absorb fat droplets [122].

3.3.2.2 Receptor-mediated endocytosis

Receptor-mediated endocytosis is the best route for the uptake of certain macromolecules. Initially, macromolecules attach to specific receptors, such as clathrin-coated pits at the cell surface. The pits germinate in the membrane to form small clathrin-coated vesicles containing receptor with bonded macromolecules. Eventually, clathrin-coated vesicles merge with the primary endosome and are transferred to the lysosome or recycled to plasma membrane [128]. Fig. 6 shows the indirect penetration mechanisms.

Fig. 6. Comparison between phagocytosis, pinocytosis and receptor-mediated endocytosis. Picture taken and modified from Ref. [129].

3.4 Bioactive peptides

Generally, bioactive peptides (BP) such as ACPs are organic polymers formed by 2–20 amino acids building blocks that connected by covalent bonds, known as amide or peptide bonds [130]. Although bioactive peptides exist in natural sources, large amounts of bioactive peptides are encrypted in structure of their parent proteins. In fact, they remain inactive while the sequences are kept within the parent protein [131]. Peptide activity is evaluated by various peptide properties including the relevant spatial structure, amino acid composition, type of N and C of amino acid terminals, peptide chain length, charge, hydrophobicity and hydrophilicity of its residues. Bioactive peptides show distinct mechanisms of action in cancerous cells such as pore formation by barrel-stave model and toroidal model, Increase calcium ion entry, modification of lysosomal membrane, increase proteasome activity, induction of mitochondrial apoptotic pathway by caspase cascade, immune modulation pathway activity, and inhibition of genes relating to DNA replication with interfering with cycle cell [132]. Therapeutic peptides are divided into three categories based on the mechanism of cell entry, 1) Pore-forming peptides that bind to negatively charged molecules on the surface of cancer cells to induce apoptosis or necrosis, 2) Cell-penetrating peptides which cross the plasma membrane and transport small molecules, oligonucleotides or proteins, the action known as internalization, 3) Tumor-targeting peptides that bind to cancer cell surface receptors for cellular internalization [133]. Therapeutic peptides are also classified into three groups based on their biological targets [134,135] including: 1) Signal transduction pathways: when the signaling molecules bind to the receptor, the receptor proteins change, which initiates the transduction process involving several steps, 2) Cell cycle regulation: Cell division is a precise process vital for the production of daughter cells. The ability of a cell to divide regularly is generally attributed to the presence of two classes of molecules, cyclins and cyclin-dependent kinases (CDKs) [136], 3) Cell death pathways: There are three major types of morphologically distinct cell death patterns: apoptosis (type I cell death), autophagic cell death (type II), and necrosis (type III) [137]. In general, peptides are effective and selective signaling molecules that connect to the surface of cells and through which, can activate receptors such as G protein-coupled receptors (GPCRs) [138].

Read full article

URL: https://www.sciencedirect.com/science/article/pii/S0009279722003994

4 The state of polypeptide drug development

Peptides have many crucial functions in the human body. More than 7000 naturally occurring peptides have been identified that have critical roles in human physiology. They act as hormones, neurotransmitters, growth factors, and antibacterial agents, among others. Peptides have been widely used in many fields such as medicine, food, health care products, cosmetics, biomaterials, and biopesticides. They are mainly used in medicine as peptide drugs, peptide vaccines, and peptide diagnostic reagents [31,32].

4.1 Market applications of polypeptide drugs

According to the Global Peptide Therapeutics Market & Clinical Pipeline Insight 2026 report, published in Jan. 2020, peptides have emerged as an important class of therapeutic molecules developed by various pharmaceutical and biotechnology companies to enable targeted drug delivery for a wide range of diseases [33]. Currently, 197 peptide drugs are available commercially. Research on new peptide drugs is continuing steadily, and more than 800 peptide drugs are in the clinical pipeline. By 2026, the global market size of peptide drugs is estimated to exceed $60 billion [34].

Peptide drugs combine the advantages of small-molecule drugs and large-molecule protein drugs. They are suitable for treating some complex diseases that are difficult to cure with traditional small-molecule drugs. Advantages of peptide drugs are:

Peptide drugs have high safety and high target affinity. They are mainly eliminated through proteolytic degradation and renal filtration. The products of peptide hydrolysis are amino acids. Therefore, the metabolites of peptide drugs are generally not considered toxic.

The probability of receiving marketing approval through clinical trials is two times higher for peptide drugs than for small-molecule drugs, and the average research and development cycle for peptide drugs is 0.7 years shorter.

Compared with recombinant protein synthesis, peptide chemical synthesis technology is mature. Peptide products can easily be separated from impurities or by-products, they are of high purity, and they can easily be combined with unnatural amino acids. By contrast, the quality, purity, and yield of recombinant proteins cannot be completely guaranteed. Unnatural amino acids cannot be introduced into recombinant proteins, and recombinant proteins cannot be amidated at the ends. Moreover, the production cycle of recombinant proteins is long and the cost is high. Peptide synthesis is generally less expensive than protein and antibody drug production and more expensive than synthesizing most small-molecule drugs.

Peptide drug development has also extended to seven major disease treatment fields: rare diseases, tumor, diabetes, gastrointestinal disease, orthopedics, immune disorders, and cardiovascular disease. Among them, rare diseases, tumor, and diabetes are the troika driving the peptide drug market. The remaining four fields also contain a good number of blockbuster drugs, including liraglutide, dulaglutide, semaglutide, leuprolide, teriparatide, octreotide, and exenatide [35]. Representative drugs in major therapeutic areas are described in detail in the following chapters.

Furthermore, peptide drugs have been extended to orphan drugs. For example, teduglutide, a glucagon-like peptide 2 (GLP-2) receptor agonist, received marketing approval for the treatment of short bowel syndrome. Several peptides are in clinical trials in the areas of infectious diseases and inflammation. Peptides have also been used to synthesize vaccines, which have been considered an alternative to traditional vaccines to solve the possible side effects of heterogeneous multicomponent vaccines.

4.2 Polypeptide drug technology innovations

As part of basic research development, an increasing number of peptide-based drug targets or receptors have been developed for application. Natural peptides are usually unsuitable for direct use as simple therapies because of their inherent disadvantages, such as poor chemical and physical stability and a short circulating plasma half-life. With continuous innovations in peptide drug molecular design technology and peptide drug delivery technology, the main problems that had limited the clinical effect of peptide drugs (such as difficulty in oral administration, a short half-life, and poor cell membrane permeability) are continuously being resolved [36,37].

4.2.1 Product innovations

From the perspective of product innovation, multifunctional peptides, peptide-coupled drugs, cell-penetrating peptides, and other technologies have become directions of global research and development.

Multifunctional peptides couple multiple functional sites of peptides, so they can have various functions while improving the effectiveness of indications and medicinal properties. Compared with single peptides, the simultaneous activation of different signaling mechanisms with multifunctional peptides maximizes the bioactive benefits, minimizes side effects, and provides a more balanced profile of the pharmacokinetic action. Most studies are conducted by large pharmaceutical companies, including Eli Lilly, Merck, Johnson & Johnson, Sanofi, and Novo Nordisk.

Peptide–drug couplings (PDCs) are a type of conjugation drugs. In PDCs, cell-targeted peptides are conjugated with drug molecules to enhance drug targeting, improve effectiveness, and reduce adverse reactions. In contrast to antibody–drug conjugates, PDCs targeting tumor cells are composed of a peptide chain containing approximately 10 amino acids and do not induce an immune response. They can be prepared at a large scale using the SPSS method. PDCs are quickly eliminated by the kidneys and are less toxic to the bone marrow and liver. Unlike bacteriophages, adenoviruses, or other microorganisms used to transport drugs, this carrier does not contain infectious materials. Companies in this field with rapid progress internationally include Oncopeptides, Bicycle Therapeutics, Cybrexa Therapeutics, and Peptidream.

Cell-penetrating peptides (CPPs) are a class of peptides usually composed of no more than 30 amino acid residues that can directly cross the cell membrane to enter the cell. CPPs are positively charged peptide fragments of varying lengths and are rich in basic amino acid residues such as arginine and lysine. All of their secondary structures have the spatial conformation of an α-helix. They can effectively promote the transdermal absorption of biomolecular drugs and have significant value in targeted formulations, transdermal drug delivery formulations, and cosmetic fields. Thus, they significantly improve drug bioavailability.

A peptide vaccine is a type of vaccine prepared through chemical synthesis according to the amino acid sequence of some known or predicted epitope of the pathogen antigen gene. A tumor therapeutic peptide vaccine is an important research directions. A myriad of differences exist between tumor cells and normal cells; however, the most fundamental difference is in tumor cell mutation. If specific neoantigens can be found in tumor cells and cancer vaccines can be developed based on the amino acid sequence of these neoantigens, the relevant immune cells will be activated to kill tumor cells with the same antigen. Current challenges are poor immunogenicity and human leukocyte antigen (HLA) phenotypic limitations on efficacy. A prophylactic peptide vaccine is an essential emerging field.

4.2.2 Delivery method innovations

From the perspective of drug delivery innovation, low oral bioavailability may be caused by the unstable structure of peptide molecules in the digestive tract, which can be hydrolyzed rapidly, the first metabolic effect of the liver, and the possibility of forming polymers. Problems such as stability can be solved by changing the modification process, whereas the biggest obstacle to peptide drug development is the inability to administer these drugs orally. In general, various proteolytic enzymes in organisms can rapidly degrade peptide drugs. Thus, peptide drugs have a poor oral effect and short half-life. Peptide drugs are administered in a relatively simple manner, mainly through intravenous injection or a drip, and approximately 75% peptide drugs are administered through injection.

Researchers are attempting to optimize the stability of the peptide structure by modifying this structure and adding barriers. Peptide structure modification increases the stability of peptides and reduces the frequency of injection administration by modifying the peptide into a cyclic form, inserting unnatural amino acids, replacing individual amino acids, and coupling structures such as polyethylene glycols, lipids, and proteins.

Regarding the injectable drug delivery system, a polymer material can be used to wrap the peptide, blocking contact between the peptide and protease to protect the peptide. Moreover, studies have investigated including peptides in liposomes for sustained release through intravenous injection.

Combining peptide drugs with osmotic agents can increase the permeability of the intestinal mucosa, allowing them to pass the intestinal mucosa quickly and be absorbed into the blood. However, the oral effect is generally lower than the injection effect. The approval of Novo Nordisk's oral Sommarutide in 2019 shook the market. However, clinical data showed that the bioavailability of that drug was only approximately 1%. Furthermore, various restrictions had to be imposed when the drug was administered. Nevertheless, with the continuous breakthrough of research, oral peptide drugs are still a significant direction for research and development.

Drugs with high efficacy and low toxicity are the eternal pursuit of humans. Compared with common drugs, peptide drugs have a longer life cycle, and are a promising field. With the continuous upgrading and iteration of global gene technology, information technology, omics technology, disease biology, and other technologies, the peptide industry has gradually changed from the classical peptide industry to the peptide economy industry. For the peptide economy industry, multifunctional peptides, conjugate peptides, binding peptides, oral peptides, gene vectors, and peptide vaccines will become the driving force of development. People's enthusiasm for peptide drugs also reached a climax with the upsurge of the market.

Read full chapter

URL: https://www.sciencedirect.com/science/article/pii/B9780443186110000243

Chapters and Articles

Peptides

Antimicrobial peptides

Nature-derived peptides

Peptides

Physicochemical Nature of Peptides

Prediction of Electrophoretic Mobility of Peptides

Electrophoretic Systems – Separation Strategies

Detection Techniques

1 Introduction

7.08.2 Peptides/Proteins

7.08.2.1 Primary Structures

7.08.2.2 Secondary Structures

7.08.2.3 Tertiary Structures

7.08.2.4 Stability of Protein Folds

7.08.2.5 Peptides as Building Blocks for Hybrid Materials

7.08.2.6 The Promise of Coiled-Coils

1 Introduction

5.4 Polyethylene Glycol-Peptide Conjugation

Physicochemical Nature of Peptides

3 Design of peptide-based probes for PET imaging

3 Properties of peptides

3.1 Structure of peptides and proteins

3.2 Chemical bonds that play vital roles in 3D structure of peptide/protein

3.3 Peptide entry into cell

3.4 Bioactive peptides

4 The state of polypeptide drug development

4.1 Market applications of polypeptide drugs

4.2 Polypeptide drug technology innovations

Peptide Therapy Guide Editorial Team

Biochemistry, Peptide - StatPearls - NCBI Bookshelf - NIH

Explainer: Peptides vs proteins - what's the difference?

Peptides: Types, Uses, and Benefits