Peptide ligand-mediated targeted drug delivery of nanomedicines

Zhuxuan Jiang a, Juan Guan a, Jun Qian b and Changyou Zhan *ab
aDepartment of Pharmacology, School of Basic Medical Sciences & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 200032, P.R. China. E-mail:; Tel: +86-21-54237379
bSchool of Pharmacy, Fudan University & Key Laboratory of Smart Drug Delivery (Fudan University), Ministry of Education, Shanghai 201203, P.R. China

Received 23rd October 2018 , Accepted 31st December 2018

First published on 18th January 2019

Targeted drug delivery is emerging as a promising strategy to achieve better clinical outcomes. Actively targeted drug delivery that utilizes overexpressed receptors or antigens on diseased tissues is receiving increasing scrutiny, especially due to the uncertainty of existence of the enhanced permeability and retention (EPR) effect in cancer patients. Peptide ligands are advantageous over other classes of targeting ligands due to their accessibility of high-throughput screening, ease of synthesis, high specificity and affinity, etc. In this review, we briefly summarize the resources of peptide ligands and discuss the pitfalls and perspectives of peptide ligand-mediated targeted delivery of nanomedicines.

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Zhuxuan Jiang

Zhuxuan Jiang received his B.S. from Fudan University in 2017. Currently, he is a Ph.D. student supervised by Prof. Changyou Zhan in the Department of Pharmacology, School of Basic Medical Sciences of Fudan University. His research focuses on the development of targeted drug delivery systems.

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Juan Guan

Guan Juan received her B.S. from Taishan Medical University in 2013 and her M.S. degree in Pharmacology from Shandong University in 2016. Currently, she is a Ph.D. student supervised by Prof. Changyou Zhan in the Department of Pharmacology, School of Basic Medical Sciences of Fudan University. Her research interests focus on the mechanistic understanding of targeted drug delivery.

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Jun Qian

Dr Jun Qian is an associate professor at the School of Pharmacy, Fudan University. She received her Ph.D. in Pharmaceutics in 2014 from the School of Pharmacy, Fudan University. Dr Qian was appointed as the associate director of the Department of Radiopharmacy at the School of Pharmacy, Fudan University in 2014. Her research centers on the biomedical applications of radionuclide tracers and the development of nanoprobes for brain tumor imaging.

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Changyou Zhan

Dr Changyou Zhan is a professor of pharmacology at the School of Basic Medical Sciences, Fudan University. He obtained his Ph.D. in Pharmaceutics from Fudan University in 2010. His recent research focuses on understanding the in vivo delivery mechanism of liposome-based targeted drug delivery systems.

1. Introduction

In the past few decades, targeted drug delivery has emerged as a promising method to achieve better treatment by enhancing drug accumulation in lesions and/or by decreasing side effects. Angiogenesis frequently occurs in solid tumors and leaky vasculatures form, allowing nanocarriers to accumulate in the tumor region by passive targeting.1,2 In contrast, active targeting centers on recognition of overexpressed receptors or antigens by modifying specific ligands on the surfaces of nanocarriers. Numerous ligands have been exploited for targeted delivery, including antibodies, aptamers, small molecules, and peptides (Fig. 1).
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Fig. 1 Summary of different classes of ligands exploited for targeted drug delivery.

1.1 Antibodies

Antibodies and their fragments have been extensively exploited for targeted drug delivery due to their high specificity and affinity for cognate antigens.3–5 Antibodies or antibody fragments can be modified on the surfaces of nanocarriers such as liposomes, a class of vehicles known as immunoliposomes, for targeted drug delivery. Immunoliposomes can achieve higher drug loading capacity by carrying payloads using lipid bilayers; however, there is a lack of reliable chemistry to attach antibodies on the nanocarriers.6,7 Additionally, the potential immunogenicity of antibodies and fragments is a major challenge that may severely restrict their clinical applications. Fc fragments can direct fast clearance of liposomes by activating mononuclear phagocytic systems.8

1.2 Aptamers

Aptamers are a class of oligonucleotides that can bind to cognate receptors with high specificity and affinity. The chemical properties of aptamers provide superior permeability and solubility and lower immunogenicity.9 Aptamers can be directly modified on the surfaces of nanocarriers, including liposomes, dendrimers and polymeric nanoparticles (reviewed by ref. 10). However, unmodified aptamers exhibit short half-lives due to high clearance rates and rapid degradation by nucleases.11 Additionally, the specificity of aptamers relies on their secondary and tertiary conformations; these can be severely affected by various factors, such as temperature, pH and ionic strength.9,10 Slight changes in the microenvironments of target sites may exert great influences on aptamer–receptor interactions.

1.3 Small molecules

Small molecule ligands provide potent options for targeted drug delivery. For example, folic acid (FA), also known as vitamin B9, is necessary for DNA synthesis, metabolism and repair.12 Meanwhile, cancer cells overexpress folate receptors (FRs) with high folic acid binding affinity.13 FA-modified anti-cancer targeted drug delivery systems have gained increasing scrutiny, including FA-conjugated drugs,14 liposomes15,16 and other nanocarriers.17,18 They are generally less immunogenic and are easy to synthesize. However, the limited binding interfaces between small molecules and receptors restrict their binding specificity. Many of their receptors are nonspecifically expressed on tumor cells; thus, they may be toxic to normal cells.19,20 Additionally, chemical conjugation with drugs or nanocarriers can dramatically decrease the binding affinity of small molecule ligands.

1.4 Peptides

Peptide ligands have sizes that fall between those of small molecule ligands and antibody ligands. Peptide ligands can simulate protein–protein interactions and have large binding interfaces with receptors; thus, they possess much higher binding affinity and specificity than small molecule ligands. Peptides offer a potent resource for targeted drug delivery. Compared to protein ligands, peptides have many advantages, including better penetration, ease of synthesis, and lower immunogenicity and cost. Large scale synthesis of peptides presents a convenient and economical option for drug use; also, due to the abundant chemical groups in peptides, they are suitable for manipulation. This review will briefly introduce methods to design or screen new peptide ligands for targeted delivery and will discuss the remaining challenges in this field.

2. Peptide ligands for targeted delivery

Peptide ligands used for targeted drug delivery are mainly identified via bio-inspired techniques (biomimetic peptides) or large scale screening of peptide libraries (such as phage display peptide libraries and chemical peptide libraries, Table 1). In particular, most peptide ligands are exploited to target cancer cells and/or penetrate biological barriers (such as the blood–brain barrier and cell membranes). These peptide ligands have a great variety of origins, structures, targets and biomedical applications, providing vast resources for achieving targeted drug delivery.
Table 1 Summary of peptide ligands for targeted delivery of nanomedicines
Peptide Structure Sequencea,b Receptor Application Ref.
a Cysteine residues that form disulfide bonds are indicated in bold and italic. b D-Amino acids are indicated in lowercase letters.
Octreotide Cyclic (disulfide bond) fCFwKTC-Thr(ol) Somatostatin receptors Carcinoid syndrome, glucagonoma, and gastrinoma targeting 25 and 28–32
Bombesin Linear Pyr-QRLGNQWAVGHLM-NH2 GCP receptors GCP receptor-positive cancer targeting 33–35
KC2S Cyclic (disulfide bond) YTKTWCDGFCSSRGKRIDLG nAChRs Brain targeting 44
CDX peptide Linear FKESWREARGTRIERG nAChRs Brain targeting 45, 46, 105 and 115
LyP-1 peptide Cyclic (disulfide bond) CGNKRTRGC p32/gC1qR Tumor cells, tumor lymphatics and tumor-associated macrophage targeting 49–51 and 99
SP94 Linear SFSIIHTPILPL Unknown receptor on hepatocellular carcinoma cells Hepatocellular carcinoma targeting 52–55
A7R Linear ATWLPPR VEGFR-2; NRP-1 Glioma, glioma vasculogenic mimicry and neovasculature targeting 56–59
c(RGDfV) Cyclic (head to end amide reactions) Cyclo(RGDfV) Integrin αvβ3 RGD peptide with improved stability 70–75
iRGD Cyclic (disulfide bond) CRGDKGPDC Integrin αvβ35 RGD peptide with enhanced cell-penetrating ability 76 and 77
OV02 Cyclic (disulfide bond) cdG-HCit-GPQc Integrin α3 Ovarian cancer targeting 88 and 89
PLZ4 Cyclic (disulfide bond) cQDGRMGFc Unknown receptor on bladder cancer cells Bladder cancer targeting 90–92
Angiopep-2 Linear TFFYGGSRGKRNNFKTEEYC LRP-1 Brain targeting 102–104
TK Linear TWYKIAFQRNRK α6β1 Colon cancer targeting 125 and 126

2.1 Biomimetic peptides

Understanding of biological processes in nature has expanded remarkably over the past decades. For example, numerous receptors that are involved in the development and progress of cancers have been found to interact with naturally occurring proteins or peptide toxins, which provides a starting point for the identification and design of biomimetic peptides to achieve targeted drug delivery (Fig. 2).21–23 Many naturally occurring proteins and peptides are potent ligands for their receptors; however, their direct applications for targeted delivery are restricted by various issues, such as low biocompatibility, poor specificity, high toxicity and large size. Based on our understanding of these templates, structure-based peptide optimization can be conducted to identify biomimetic peptide ligands that can overcome the shortcomings of their templates and provide advantages such as high stability, enhanced specificity and affinity, and lack of toxicity.
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Fig. 2 Development of biomimetic peptide ligands for targeted drug delivery. Naturally occurring protein toxins are starting points for the development of biomimetic peptide ligands with short sequences, high affinities and stabilities, and lack of toxicity via structure-based peptide design and/or computer-aided peptide design. The resulting peptide ligands can facilitate targeted delivery of nanocarriers after modification of their surfaces.

Somatostatin was first isolated in the 1970s as a peptide acting on the pituitary gland.24 In the 1980s, rational design and optimization was conducted based on the minimal required fragment in somatostatin, leading to the discovery of SMS 201-995 (also known as octreotide).25 Five subtypes of somatostatin receptors are expressed on normal cells; these are up-regulated in many types of cancers.26,27 Therefore, somatostatin analogs are ideal tools for targeted anti-cancer therapy. For example, octreotide has been applied for targeted delivery of radiotherapeutic agents,28 chemotherapeutic agents,29 liposomes30,31 and micelles.32

Many natural peptides provide templates for biomimetic design even if they are non-human derived. Bombesin, a tetradecapeptide isolated from the European frog bombina in 1970,33 has the same functional domain as some human hormones, such as neuromedin B (NMB) and gastric-releasing peptide (GCP), which contain highly conserved C-terminal sequences.34 GCP receptors are overexpressed in some types of cancer cells; thus, bombesin and its analogs can facilitate targeted delivery to GCP receptor-positive cancers. A bombesin analog (BN7-14) conjugated with doxorubicin-loaded liposomes demonstrated in vivo PC-3 xenograft homing effects and enhanced anti-tumor efficacy.35

Pathogens also provide ideal resources for biomimetic design of peptide ligands. Kumar et al. successfully designed a 29-mer peptide (RVG-29) that can specifically bind nicotinic acetylcholine receptors (nAChRs) and cross the blood–brain barrier (BBB) by mimicking the central nervous system (CNS) targeting motif of rabies virus glycoprotein.36 RVG-29 could complex with siRNA and facilitate brain transport after addition of a nine-arginine sequence.37,38 RVG-29 modification presents a potent approach for brain targeting in many nano-sized drug delivery systems, including liposomes, PLGA nanoparticles,39 chitosan-conjugated pluronic-based nanocarriers40 and silica-coated gold nanorods.41,42

The sequences of many protein toxins are long; thus, their structures are too large for drug use. Efforts to optimize toxin templates should be made to minimize size and decrease toxicity while maintaining the specific ligand activity. In recent years, many such efforts have been made, and several toxin-derived biomimetic peptides have been exploited for targeted drug delivery (reviewed by ref. 43). Zhan et al. acquired a biomimetic peptide (KC2S) that can specifically bind to nAChRs with high affinity by mimicking the loop 2 segment of Ophiophagus hannah toxin b (KC2S); this peptide exhibited potent brain targeting ability when modified on the surface of PEG-PLA micelles.44 However, the disulfide bond in KC2S that is necessary for receptor binding is prone to reduction in blood. A shorter peptide without this disulfide bond for recognition of nAChRs, known as CDX, was identified by computer-aided peptide design. CDX peptide is a biomimetic peptide derived from candoxin, a three-finger snake neurotoxin isolated from Bungarus candidus.45 CDX modification dramatically improved the anti-glioma effects of PTX-loaded micelles in nude mice.46 Based on a similar principle, another toxin-mimicking brain-targeting peptide was developed based on the template of bee venom apamin. Oller-Salvia et al. designed a Mini-Ap-4 peptide that can effectively achieve targeted brain delivery.47

2.2 Phage display-screened peptides

Since it was first described in 1985,48 phase display has evolved into a widely used method for high-throughput screening of peptides. By ligating exogenous DNA into phage gene-encoding coat proteins, proteins or peptides of interest can be encoded and expressed along with coat proteins on the phage surface. A library of peptides can be displayed on phage surfaces by introducing a DNA library consisting of randomized sequences. In the past few decades, phage display has become a major technique to develop new ligands, and numerous peptides have been acquired for targeted delivery (Fig. 3).
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Fig. 3 Identification of peptide ligands by phage or OBOC libraries using receptors, cells or in vivo screening.

Unlike structure-based biomimetic design, phage display provides a method to develop peptide ligands without knowledge of binding information; this has led to a rapid increase in new peptide ligands. LyP-1 is a peptide that can specifically bind tumor cells, tumor lymphatics and tumor-associated macrophages. The receptor for LyP-1 binding was unclear when it was first discovered by in vivo phage display.49 Fogal et al. found that the protein p32 (also named gC1q) functions as the receptor of LyP-1.134 LyP-1 peptide presents a potent ligand for anti-cancer targeted drug delivery systems, including liposomes and nanoparticles.50,51

In vitro phage display is more convenient in comparison to in vivo phage display and can be used for ligand screening of designated cells. Likewise, when utilizing cells for phage display, the receptors may be unknown. For example, Lo et al. identified a novel peptide ligand (SP94) using in vitro phage display; the peptide bound specifically to unknown receptors on hepatocellular carcinoma cells.52 Modification of SP94 successfully guided drug-carriers into tumors to achieve targeted delivery.53–55

Phage display also presents an efficient platform for screening peptide ligands of designated receptors. Vascular endothelial growth factor (VEGF) can bind the kinase domain receptor (KDR/FLK1 or VEGFR-2) to initiate vascularization and tumor-induced angiogenesis; thus, VEGF-KDR is a potential target for cancer therapy. Binetruy-Tournaire et al. screened a peptide, termed A7R, using membrane KDR and VEGF-antibody.56 A7R has been applied in many anti-cancer drug delivery systems as a tumor-targeting ligand.56–58 Aided by computer-aided peptide design, Ying et al. reported a cyclic A7R peptide that possessed higher affinity and stability than the linear A7R peptide.59

Several peptide motifs have been discovered using phage display in the past decades. These motifs are the minimal recognition segments for receptor binding. NGR and RGD are among the most studied motifs. NGR, a peptide motif selectively homing tumor vasculature, was originally discovered by in vivo phage display.60 Subsequently, aminopeptidase N (CD13) on the surface of a wide range of cancer cells was identified as the receptor of NGR peptides.61 Both linear and cyclic peptides containing the NGR motif have been revealed to direct tumor-targeted delivery of liposomes62–64 and nanoparticles.65,66

RGD is another popular motif for cancer targeting. Both biomimetic design and phage display have been utilized, along with many assistive techniques such as computer-aided peptide design, to develop the RGD family. RGD was first identified as a cell recognition segment from fibrinogen67 by binding integrins with high specificity and affinity. Integrin αvβ3 is widely overexpressed in many types of cancers and is involved in tumorigenesis; thus, the RGD peptide family have emerged as potent peptide ligand resources for tumor targeting.68,69 Among the RGD peptide family, cyclic RGD peptides tend to be more potent and stable than linear peptides. Based on the structure–activity relationships between RGD and integrin αvβ3, a cyclic RGD peptide with high affinity was designed, known as c(RGDfV), which potently directs tumor-targeted delivery of micelles,71,72 PLGA nanoparticles73 and liposomes.74,75 iRGD, a peptide ligand that binds integrins and penetrates deeply into tumor tissues, arguably represents major progress in the development of the RGD peptide family. The design of iRGD was inspired by the discovery of another peptide motif that can bind neuropilin-1 (NRP-1), a co-receptor of VEGF. This motif (R/KXXR/K), named CendR, can facilitate intracellular endocytosis and trans-tissue transportation.76 iRGD conjugation with drug delivery systems has significantly enhanced drug accumulation in and penetration into tumors.77 Because numerous RGD peptides have been explored and many reviews have been published,78–80 we will not expand the description of the RGD peptide family here.

2.3 Peptides screened from chemical peptide libraries

Peptide screening from chemical libraries provides a promising way to obtain new peptide ligands. The one-bead one-compound (OBOC) peptide library, first developed by Lam et al. in 1991, is the most commonly used technique in this field.81 By applying a “split-mix” strategy to solid phase peptide synthesis, OBOC libraries can be acquired where each compound bead displays one peptide for further striping and decoding (Fig. 3). Several peptide ligands screened from OBOC libraries have been applied for targeted delivery of nanomedicines. For example, Jia et al. reported an OBOC library-derived peptide that specifically bound to CD13; liposomes functionalized with the peptide can achieve efficient targeted drug delivery to CD13-overexpressing tumors in vitro and in vivo.82

In contrast to phage display, the OBOC technique is limited to in vitro screening. On the other hand, it grants the ability to build more diversified peptide libraries (reviewed in ref. 83 and 84). While phage display libraries only comprise natural L-amino acid sequences with simple configurations, OBOC libraries offer the option to employ unnatural amino acids and provide more structural possibilities, such as cyclic, branched and macrocyclic peptide libraries (reviewed in ref. 85). For example, OBOC libraries are suitable for the discovery and optimization of cyclic RGD sequences containing D-amino acids.86,87 When OBOC libraries were designed based on another original integrin-binding cyclic peptide motif, a novel peptide, OV02, was identified which has high specificity and affinity to α3 integrin overexpressed on ovarian cancer cells.88 OV02-PEG modified nanoparticles were potent carriers for targeted drug delivery against ovarian cancer.89 Similarly, Zhang et al. used combinatorial cyclic peptide libraries and screened out a cyclic peptide named PLZ4, which selectively bound to bladder cancer cells in vitro and in vivo.90 Further research indicated that PLZ4-modified micelles loaded with chemotherapeutics could achieve efficient targeted drug delivery against bladder cancer.91,92 Aided by computational design, Geng et al. reported a HER2 peptide ligand acquired from OBOC peptide libraries to improve the targeted delivery of DOX-loaded liposomes against HER2-positive cells.93

There are several other approaches for exploring new peptide ligands, such as PNA-encoded solution phase peptide libraries (reviewed in ref. 94 and 95) and peptide microarrays (reviewed in ref. 94 and 96). These methods offer additional libraries for peptide screening; however, to date, their applications in targeted drug delivery of nanomedicines have been rare.

3. Pitfalls of peptide ligand-mediated targeted drug delivery

Despite their many advantages over other ligands, the application of peptides for targeted drug delivery is impaired by pitfalls such as poor stability and unexpected immunogenicity, which have become key problems in this field. Any breakthrough to overcome these pitfalls will benefit the future development of targeted drug delivery systems.

3.1 Stability

A major problem of peptide ligands is their poor stability. As “guides” of therapeutic agents, the targeting ligands must remain stable and active for receptor binding. An unstable ligand may lose bioactivity, leading to off-targeting of drug delivery systems. Peptides are usually sensitive to enzymatic microenvironments (such as in blood circulation and lysosomes) and are readily degradable, which severely restricts their applications for targeted drug delivery.

Cyclization is a classic method to stabilize peptides against proteolysis. Peptide cyclization is achievable by the formation of disulfide bonds, amide bonds or other chemical bonds. The aforementioned cyclic RGDfV is a typical head-to-tail cyclic peptide using amide bonds. Compared to linear RGD analogs, cyclic RGDfV presents much higher activity and stability.70–72 In addition, stapled peptides have demonstrated enhanced stability. The stapled peptide technique is a promising method to stabilize α-helical peptides by applying ring-closing reactions to α-methyl-substituted amino acids, thus forming synthetic braces.97 Ruan et al. designed a stapled RGD peptide and proved it to be a potent ligand to enhance BBB penetration while maintaining the ability to target glioma cells.98 Similarly, the cyclic peptide c(LyP-1) by altering the disulfide bond in LyP-1 to amide bond exhibited high stability compared to the LyP-1.99

The development of peptidomimetics is also a useful method to design stable peptides using unnatural amino acids as building blocks. D-Peptides (either all-D or partial-D peptides) have become remarkably predominant. In addition to the OBOC library screening technique, there are two main approaches to obtain D-peptides: retro-inverso isomerization and mirror-image phage display (reviewed in ref. 100).

Retro-inverso isomerization was first introduced as a method to acquire D-peptides by Goodman et al. in 1979.101 Based on a known sequence, a new peptide is designed with all D-amino acids assembled in the reverse order to the original L-peptide. In the ideal situation, D-peptides possess similar side chain topologies and biological activities to their original L-peptides but have much higher stability.100 Angiopep-2, for example, is a peptide ligand for LRP-1, which is overexpressed on brain capillary endothelial cells. Angiopep-2-modified nanocarriers achieved significantly increased brain distribution.102,103 Wei et al. used the retro-inverso isomer of Angiopep-2, named DAngiopep-2, to establish a brain-targeted drug delivery system. Although DAngiopep-2 demonstrated relatively lower uptake efficiency by brain capillary endothelial cells than LAngiopep-2 in vitro, it exhibited enhanced stability, and the modified micelles displayed higher distribution in normal brain and intracranial glioblastoma cells than LAngiopep-modified micelles.104DCDX, the retro-inverso isomer of LCDX peptide, was also identified as a potent brain-targeting ligand when modified on DOX-loaded liposomes.105 Giralt and coworkers obtained a full protease-resistant, 12-mer peptide with the capacity to act as a BBB shuttle by applying the retro-inverso approach to a peptide that targets the transferrin receptor. This peptide could enable the transport of a variety of cargos into the central nervous system.106 Notably, not all retro-inverso peptides can maintain the biological activities of their original L-peptides. Li et al. investigated four peptides and their retro-inverso forms with different receptors; they found that some retro-inverso peptides, such as Dp53(15–29), exhibited significantly lower binding activities than their original L-peptides.107 This may be related to their structure–activity relationships, because all four of these ligand–receptor binding sites require α-helix structures of original L-peptide ligands; this suggests that the retro-inverso isomerization technique should not be applied for L-peptides containing α-helices.

While retro-inverso isomers are available for known sequences and can be applied for uncertain receptors, mirror-image phage display is an approach to screen D-peptides for known receptors without L-peptide templates. To accomplish mirror-image phage display, a D-form target protein must be chemically synthesized which has the same sequence as the target protein, yet is composed of D-amino acids. After successfully screening out L-peptides with high affinity, the D-peptides can be acquired by simply converting the L-amino acids to their D-forms. Since this technique was first developed in 1996,108 many D-peptides have been screened for a variety of applications, such as HIV-inhibitor and Aβ(1–42) ligands (reviewed in ref. 109), using mirror-image phage display. Li et al. reported a D-peptide ligand obtained by mirror-image phage display which targets Fn14, a cell surface receptor that is overexpressed in many kinds of cancers, such as pancreatic and triple-negative breast cancers. This D-peptide represents a potent ligand to facilitate drug accumulation in tumor regions after modification on the surface of PTX-loaded liposomes.110

3.2 Immunocompatibility

It remains crucial to thoroughly understand the impact of peptides on the interactions occurring at the interface between peptide-modified nanocarriers and biological systems. The charged amino acids in peptide ligands affect the zeta potential after being modified on the surface of nanomedicines. Positively charged peptide ligands may interact with cells directly and disturb the continuity of cell membranes by electrostatic interactions, leading to damage to biological barriers and toxicity to tissues and organs.105,111 The positively charged ligands exposed on nanomedicines can be ultimately digested by lysosomes.112 This procedure presents antigens to T cells and activates the cellular immune response. Additionally, mononuclear phagocytic systems tend to engulf positively charged ligand-modified nanomedicines, causing an increase of mRNA expression of the inflammatory cytokine IL-6 and activation of the innate immune system.111

After entry into the bloodstream, peptide ligand-modified nanomedicines are immediately surrounded by a large number of plasma proteins, which form a protein shell called the “protein corona” (PC).113 Peptides alone are often too small to evoke a strong organism body response. However, peptides affect the absorption of the protein corona after conjugation with nanomedicines, which may be directly related to the immunocompatibility of nanomedicines, including pharmacokinetics, biodistribution, immunogenicity, toxicity and even the targeting yield. For positively charged peptide ligands, proteins absorbed on the nanoparticle surface may neutralize the positive charges after entry into the bloodstream. PCs around the nanoparticles form a protecting barrier between the peptide ligand and cell membrane. However, the absorption of proteins on the nanoparticle surface may induce conformational changes and denaturation by decreasing their terminal stability,114 which can potentially increase immunogenicity because the nanoparticles expose protein epitopes on their surfaces in aberrant conformations. The stability and length of peptide ligands are their other important properties. Peptide ligands can be readily degraded by hydrolytic enzymes in plasma. Stabilized peptide ligands can be more efficient than their L-counterparts.105 However, stable, positively charged peptide ligands may play double-edged roles after being modified on the surfaces of nanomedicines. Long peptide ligands with high stability and heavy positive charges are prone to be taken up by dendritic cells and lymph node-resident antigen-presenting cells (APCs) and generate strong immunogenicity after modification on the surface of nanomedicines.115

Thus, peptide ligands can affect the immunocompatibility of nanomedicines either by their direct interactions with immune systems or by determining the compositions of the formed PCs. For example, modification with peptide ligands can increase the absorption of complements or immunoglobulins, which subsequently activate the mononuclear phagocytic system and severely decrease immunocompatibility.116–118 It is crucial to understand the direct and indirect interactions between peptide ligand-mediated targeted drug delivery systems and immune systems.

4. Perspectives

During the past several decades, nanomedicines have received increasing scrutiny due to their advantages over traditional formulations (e.g. increasing the solubility of hydrophobic therapeutic agents and decreasing their toxicity) as well as their potential to achieve targeted drug delivery. However, clinical translation of nanomedicines is faced with unprecedented challenges. As the first FDA-approved nanomedicine, Doxil demonstrates satisfactory effects in decreasing the myocardial toxicity of doxorubicin.119–121 The clinical outcome of Doxil has been challenged recently. For passive targeting drug delivery systems, the EPR effect plays crucial roles in enhancing the tumor accumulation of payloads. Although the EPR effect in animal models has been extensively studied, the EPR effect in human cancer patients is complicated. It depends on the biological properties, location, size and clinical treatments of cancers. PEGylated liposomes prolong blood circulation and decrease the distribution of doxorubicin in the heart; meanwhile, their anticancer effects are not significantly elevated in comparison to free doxorubicin. In particular, the use of liposomes causes additional side effects, such as swelling of the feet and lower legs.122–124

Many new peptide-mediated drug delivery systems have been published recently, extending this field to additional applications. Some peptides designed many years ago have been found to be potential agents against other targets.125 For example, TK, a ligand for integrin α6β1 which was first designed to prevent melanoma invasion, was recently identified as a potent targeting agent for colonic targeted therapy because it can increase uptake by Caco-2 cells and effectively increase the penetration of tumor spheroids.126 Phage display technique enables rapid and efficient development of peptide ligands, a large number of new peptide ligands have been developed in recent years,127–133 most of which specifically bind to receptors that are overexpressed on various types of cancer cells. In addition to the increase in the number of peptide ligands, progress has also been made in the function of peptide ligands.

Peptide ligands are advantageous over many other ligands to achieve targeted drug delivery. However, modification of peptide ligands may severely affect the stability, blood circulation, biodistribution and safety of nanomedicines. Moreover, the effects of peptide ligands may differ from those of nanomedicines. For example, the effects of peptide ligands on liposomes may be different from their effects on PLGA nanoparticles. Thus, studies on nanomedicines should be personalized. Intensive mechanistic studies should be conducted to understand how targeted nanomedicines can reach the target organs/tissues/cells. For example, bio-nano interactions in blood circulation and tissues strictly decide the fate of nanomedicines; meanwhile, peptide ligands severely affect these bio-nano interactions.115 It is anticipated that deciphering the effects of peptide ligand structures on bio-nano interactions may result in the design of intriguing nanomedicines with high clinical translational potential.

Notably, during the past decades, excessive attention has been paid to the nano-side of nanomedicines (Fig. 4). Explosive growth of our understanding of novel nanocarriers with new functions, modalities and even intelligence has been witnessed; meanwhile, the effects of therapeutic agents on nanomedicines have been overlooked. Doxorubucin and paclitaxel, which are no longer first-class chemotherapeutic agents in many clinical cases, are still being widely studied in many recent literature publications. In many cases, the dose-effect relationships must be interrogated. For example, simply increasing doxorubicin accumulation may be ineffective in the chemotherapy of many peripheral cancers. Systemic delivery of a free drug may reach the plateau of dose-effect relationships. In addition, in vivo drug release profiles that have not been extensively studied due to limitations of detection methods may be crucial. Too-slow release in cancer tissues may induce drug resistance.

image file: c8bm01340c-f4.tif
Fig. 4 Current status of research on nanomedicines. Many advances have been witnessed on the nano-side, such as new materials, functions, modalities and intelligence of nano-based delivery systems, leaving the medicine-side behind far behind.

Conflicts of interest

There are no conflicts to declare.


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These authors contributed equally to this work.

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