Cell penetrating peptides and their applications in siRNA delivery

Given its ability to knock down essentially any gene of interest, siRNA has been one of the promising candidates for gene therapy. However, like other nucleic-acid-based drugs, its poor cellular uptake poses a major challenge. Here we briefly summarize the use of cell penetrating peptides （CPPs） as a novel and promising approach for siRNA delivery. The main advantages of CPPs are their low toxicity and high efficiency.

Cationic and amphipathic cell-penetrating peptides （CPPs）： Their structures and in vivo studies in drug delivery

Over the past few decades, cell penetrating peptides （CPPs） have become an important class of drug carders for small molecules, proteins, genes and nanoparticle systems. CPPs represent a very diverse set of short peptide sequences （10-30 amino acids）, generally classified as cationic or amphipathic, with various mechanisms in cellular internalization. In this review, a more comprehensive assessment of the chemical structural characteristics, including net cationic charge, hydrophobicity and helicity was assembled for a large set of commonly used CPPs, and compared to results from numerous in vivo drug delivery studies. This detailed information can aid in the design and selection of effective CPPs for use as transport carriers in the delivery of different types of drug for therapeutic applications.

Overcoming the cellular barriers and beyond: Recent progress on cell penetrating peptide modified nanomedicine in combating physiological and pathological barriers

The complex physiological and pathological conditions form barriers against efficient drug delivery.Cell penetrating peptides(CPPs),a class of short peptides which translocate drugs across cell membranes with various mechanisms,provide feasible solutions for efficient delivery of biologically active agents to circumvent biological barriers.After years of development,the function of CPPs is beyond cell penetrating.Multifunctional CPPs with bioactivity or active targeting capacity have been designed and successfully utilized in delivery of various cargoes against tumor,myocardial ischemia,ocular posterior segment disorders,etc.In this review,we summarize recent progress in CPP-functionalized nano-drug delivery systems to overcome the physiological and pathological barriers for the applications in cardiology,ophtalmology,mucus,neurology and cancer,etc.We also highlight the prospect of clinical translation of CPP-functionalized drug delivery systems in these areas.

Protein transduction domain of membrane penetrating peptide can efficiently deliver DNA and protein into mouse liver for gene therapy

BACKGROUND: The development of a harmless and effi- cient nonviral gene delivery system that can facilitate the penetration of nucleic acids through the plasma membrane is a key to successful gene therapy. The aim of this study was to test a nonviral gene transferring vector's function of delivering DNA into liver cells to provide an important clue for gene transfer in liver gene therapy. METHODS: The complex of DNA and DNA delivering protein was injected into mice through their tail veins. Then the mice were killed and their liver tissue was sec- tioned. The gene transferring results were detected using a confocal laser scanning microscope. RESULTS: Fluorescence analysis indicated that both DNA- membrane penetrating peptide (MPP) complex and DNA- hepatocyte specific receptor binding domain ( HSRBD) - MPP complex could go into liver cells. The fluorescence value of liver cells in the DNA-HSRBD-MPP group was higher than that in the DNA-MPP group. CONCLUSIONS; MPP can successfully deliver DNA and protein into cells, and MPP with a HSRBD can specifically deliver DNA into liver cells. These have laid a foundation for further study on the nonviral liver cell gene delivering system.

Effects of grafting cell penetrate peptide and RGD on endocytosis and biological effects of Mg-CaPNPs-CKIP-1 siRNA carrier system in vitro

Calcium phosphate nanoparticles(CaPNPs)have good biocompatibility as gene carriers;however,CaPNPs typically exhibit a low transfection efficiency.Cell penetrate peptide(TAT)can increase the uptake of nanoparticles but is limited by its non-specificity.Grafting adhesion peptide adhesion peptide on carriers can enhance their targeting.The Plekho1 gene encodes casein kinase-2 interacting protein-1(CKIP-1),which can negatively regulate osteogenic differentiation.Based on the above,we produced a Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system via hydrothermal synthesis,silanization and adsorption.The effects of this carrier system on cell endocytosis and biological effects were evaluated by cell culture in vitro.The results demonstrate that CaPNPs with 7%Mg(60 nm particle size,short rod shape and good dispersion)were suitable for use as gene carriers.The carrier system boosted the endocytosis of MG63 cells and was helpful for promoting the differentiation of osteoblasts,and the dual-ligand system possessed a synergistic effect.The findings of this study show the tremendous potential of the Mg-CaPNPs-RGD-TAT-CKIP-1 siRNA carrier system for efficient delivery into cells and osteogenesis inducement.

Hepatocyte gene transfer mediated by stable polyplexes based on MPP-containing DNA complexes

BACKGROUND: In the field of gene therapy, viral vectors as delivery tools have a number of disadvantages for medical application. This study aimed to explore a novel nonviral vector as a vehicle for gene therapy. METHODS: Transvector-rpE-MPP and EGFP (enhanced green fluorescent protein) were used as the gene transfer carrier and the reporter gene, respectively. Polyplexes which integrate transvector-rpE-MPP, the object gene, and EGFP were formed. The optimal charge ratio, stability, and transduction capacity of the polyplexes in mouse hepatocytes in vitro and in mouse liver in vivo were investigated. The polyplexes of transvector-rpE-MPP and pcDNA(3)-EGFP, with charge ratios of 0, 0.25, 0.5, 0.75, 1 and 1.5 were compared to determine the optimal charge ratio. RESULTS: Polyplexes with charge ratios of 1: 1 were most stable; pcDNA(3)-EGFP in these complexes resisted digestion by DNase I and blood plasma. On the other hand, pcDNA(3)-EGFP alone was digested. Fluorescence analysis indicated that transvector-rpE-MPP successfully delivered the reporter gene EGFP into hepatocytes and that EGFP expression was detected in hepatocyte cultures and in liver tissue. CONCLUSION: These results have laid a foundation for further study of a novel nonviral gene delivery system.

Essential Gene(s) Targeted by Peptide Nucleic Acids Kills <i>Mycobacterium smegmatis</i>in Culture and in Infected Macrophages

Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.

Folate and cell penetrating peptide modification effects on penetration behavior of nanostructured lipid carriers using multicellular tumor spheroids

Surface modification may have important influences on the penetration behavior of nanoscale drug delivery system. In the present study, we mainly focused on whether cell targeting or cell penetration could affect penetration abilities of nanostructured lipid carriers（NLC）. Real--time penetration of folate--or cell penetrating peptide（CPP）-modified NLC was evaluated using a multicellular tumor spheroid（MTS） established by stacking culture method as an in vitro testing platform. The results suggested that CPP modification had a better penetration behavior both on penetration depth and intensity compared with folate-modified NLC at the early stage of penetration process.

Modification of oligonucleotides by isonucleosides incorporation and peptides conjugation

Synthetic oligonucleotides including antisense oligonucleotides and siRNA have shown promising therapeutic potential.However,to realize the therapeutic potential of synthetic oligonucleotides,many obstacles have to be overcome,such as their poor biological stability,non-specific activity and inadequate cell membrane permeability.In this paper,the achievements by Lihe Zhang＇s group in the study of isonucleotide modified oligonucleotides and oligonucleotides conjugated with cell penetrating peptides are summarized.

A detachable coating of cholesterol-anchored PEG improves tumor targeting of cell-penetrating peptide-modified liposomes

Cell-penetrating peptides(CPPs)have been widely used to enhance the membrane transloca-tion of various carriers for many years,but the non-specificity of CPPs seriously limits their utility in vivo.In this study,cholesterol-anchored,reduction-sensitive PEG(first synthesized by our laboratory)was applied to develop a co-modified liposome with improved tumor targeting.Following optimization of the formulation,the in vitro and in vivo properties of the co-modified liposome were evaluated.The co-modified liposome had a much lower cellular uptake and tumor spheroid uptake,but a much higher tumor accumulation compared to CPP-modified liposome,indicating the non-specific penetration of CPPs could be attenuated by the outer PEG coating.With the addition of exogenous reducing agent,both the in vitro and in vivo cellular uptake was markedly increased,demonstrating that the reduction-sensitive PEG coating achieved a controllable detachment from the surface of liposomes and did not affect the penetrating abilities of CPPs.The present results demonstrate that the combination of cholestervsitive PEG and CPPs is an ideal alternative for the application of CPP-modified carriers in vivo.

Overcoming oral insulin delivery barriers： application of cell penetrating peptide and silica-based nanoporous composites

Oral insulin delivery has received the most attention in insulin formulations due to its high patient compliance and, more importantly, to its potential to mimic the physiologic insulin secretion seen in non-diabetic individuals. However, oral insulin delivery has two major limitations： the enzymatic barrier that leads to rapid insulin degradation, and the mucosal barrier that limits insulin＇s bioavailability. Several approaches have been actively pursued to circumvent the enzyme barrier, with some of them receiving promising results. Yet, thus far there has been no major success in overcoming the mucosal barrier, which is the main cause in undercutting insulin＇s oral bioavailability. In this review of our group＇s research, an innovative silica-based, mucoadhesive oral insulin formulation with encapsulated-insulin/cell penetrating peptide （CPP） to overcome both enzyme and mucosal barriers is discussed, and the preliminary and convincing results to confirm the plausibility of this oral insulin delivery system are reviewed. In vitro studies demonstrated that the CPPinsulin conjugates could facilitate cellular uptake of insulin while keeping insulin＇s biologic functions intact. It was also confirmed that low molecular weight protamine （LMWP） behaves like a CPP peptide, with a cell translocation potency equivalent to that of the widely studied TAT. The mucoadhesive properties of the produced silica-chitosan composites could be controlled by varying both the pH and composition; the composite consisting of chitosan （25wt-%） and silica （75 wt-%） exhibited the greatest mucoadhesion at gastric pH. Furthermore, drugrelease from the composite network could also be regulated by altering the chitosan content. Overall, the universal applicability of those technologies could lead to development of a generic platform for oral delivery of many other bioactive compounds, especially for peptide or protein drugs which inevitably encounter the poor bioavailability issues.

Intercellular imaging by a polyarginine derived cell penetrating peptide labeled magnetic resonance contrast agent,diethylenetriamine pentaacetic acid gadolinium

Background The cellular plasma membrane represents a natural barrier to many exogenous molecules including magnetic resonance （MR） contrast agent. Cell penetrating peptide （CPP） is used to internalize proteins, peptides, and radionuclide. This study was undertaken to assess the value of a new intracellular MR contrast medium, CPP labeled diethylenetriamine pentaacetic acid gadolinium （Gd-DTPA） in molecular imaging in vitro. Methods Fluorescein-5-isothiocyanate （FITC） and Gd-DTPA respectively labeled with CPP （FITC-CPP, Gd-DTPA-CPP） were synthesized by the solid-phase method. Human hepatic cancer cell line-HepG2 was respectively stained by FITC-CPP and FITC to observe the uptake and intracellular distribution. HepG2 was respectively incubated with 100 nmol/ml Gd-DTPA-CPP for 0, 10, 30, 60 minutes, and imaged by MR for studying the relationship between the incubation time and T：W1 signal. The cytotoxicity to NIH3T3 fibroblasts cells was measured by 3-（4,5-dimethylthiazol-2-yl）- 2,5-diphenyltetrazolium bromide reduction assay （MTr）. Results The molecular weights of CPP labeled imaging agents, which were determined by MALDI mass spectrometry （FITC-CPP MW=2163.34, Gd-DTPA-CPP MW=2285.99）, were similar to the calculated molecular weights. Confocal microscopy suggested HepG2 translocated FITC-CPP in cytoplasm and nucleus independent with the incubation temperature. MR images showed HepG2 uptaken Gd-DTPA-CPP had a higher T1 weighted imaging （T1W1） signal, and that the T1W1 signal intensity was increasing in a time-dependent manner （r=0.972, P=0.001）, while the signal intensity between the cells incubated by Gd-DTPA for 60 minutes and the controlled cells was not significantly different （P=0.225）. By MTT, all concentrations from 50 nmol/ml to 200 nmol/ml had no significant （F=0.006, P=1.000） effect on cell viability of mouse NIH3T3 fibroblasts, compared with the control group. Conclusions The newly constructed CPP based on polyarginine can translocate cells by carrying FITC and MR contrast agent Gd-DTPA, and the intracellular concentrations are readily detectable by MR imaging, suggesting a new way for MR molecular imaging.

Significance and strategies in developing delivery systems for bio-macromolecular drugs

Successful development of a new drug is prohibitively expensive, and is estimated to cost approxi- mately S100-500 million US dollars for a single clinical drug. Yet, a newly developed drug can only enjoy its patent protection for 18 years, meaning that after this protected time period, any company can manufacture this product and thus the profit generated by this drug entity would reduce dramatically. Most critically, once a drug is being synthesized, its physical, chemical, and biological attri- butes such as bioavailability and in vivo pharmacokinetics are all completely fixed and cannot be changed. In principal and practice, only the application of an appro- priately designed drug delivery system （DDS） is able to overcome such limitations, and yet the cost of developing a novel drug delivery system is less than 10% of that of developing a new drug. Because of these reasons, the new trend in pharmaceutical development has already begun to shift from the single direction of developing new drugs in the past to a combined mode of developing both new drugs and innovative drug delivery systems in this century. Hence, for developing countries with relatively limited financial resources, a smart strategic move would be to focus on the development of new DDS, which has a significantly higher benefit/risk ratio when comparing to the development of a new drug. Because of the unmatched reaction efficiency and a repetitive action mode, the therapeutic activity of a single bio-macromolecular drug （e.g., protein toxins, gene products, etc.） is equivalent to about 10^6- 10^8 of that from a conventional small molecule anti-cancer agent （e.g., doxorubicin）. Hence, bio-macromolecular drugs have been recognized around the world as the future ＂drug-of-choice＂. Yet, among the 〉 10000 drugs that are currently available, only -150 of them belong to these bio- macromolecular drugs （an exceedingly low 1.2%）, reflect- ing the difficulties of utilizing these agents in clinical practice. In general, the bottleneck limitations of these bio- macromolecular drugs are two-fold： （1） the absence of a preferential action of the drug on tumor cells as opposed to normal tissues, and （2） the lack of ability to cross the tumor cell membrane. In this review, we provide strategies of how to solve these problems simultaneously and collec- tively via the development of innovative drug delivery systems. Since worldwide progress on bio-macromolecular therapeutics still remains in the infant stage and thus open for an equal-ground competition, we wish that this review would echo the desire to industrialized countries such as China to set up its strategic plan on developing delivery systems for these bio-macromolecular drugs, thereby realizing their clinical potential.

Semiconducting polymer dots with photosensitizer loading and peptide modification for enhanced cell penetration and photodynamic effect

This letter describes semiconducting polymer dots （Pdots） doped with a photosensitizer and modified with a cell penetrating peptide for photodynamic therapy （PDT）. The resulting Pdots exhibited efficient singlet oxygen （^1O2） generation mediated by intraparticle energy transfer. Experimental results indicated that the peptide-coated Pdots could promote the cellular uptake and increase the penetration efficiency in vitro, and effectively suppressed tumor growth and enhanced the photodynamic effect in vivo. Our results demonstrate that Pdots with photosensitizer loading and peptide modification hold great promise for cancer therapy.

The progress and perspective of strategies to improve tumor penetration of nanomedicines

Tumor penetration is important for effectively tumor targeting drug delivery.Recently,many researches are published to overcome the barriers that restrict tumor penetration and improve drug delivery efficiency.In the mini review,we first analyzed the barriers influence the tumor penetration,including tumor microenvironment barriers,nanoparticle properties,and interaction barriers between tumor and nanoparticles.To overcome the barrier,several strategies are developed,including modulating tumor microenvironment,changing particle size,transcytosis enabled tumor penetration,cell penetrating peptide modification and overcoming binding site barrier,which could effectively improve tumor penetration,and finally enhance tumor treatment outcome.

Improved method for synthesis of low molecular weight protamine–siRNA conjugate

RNAi technology has aroused wide public interest due to its high efficiency and specificity to treat multiple types of diseases. However, the effective delivery of siRNA remains a challenge due to its large molecular weight and strong anionic charge. Considering their remarkable functions in vivo and features that are often desired in drug delivery carriers, biomimetic systems for siRNA delivery become an effective and promising strategy. Based on this, covalent attachment of synthetic cell penetrating peptides(CPP) to siRNA has become of great interest. We developed a monomeric covalent conjugate of low molecular weight protamine(LMWP, a well-established CPP) and siRNA via a cytosol-cleavable disulfide linkage using PEG as a crosslinker. Results showed that the conjugates didn't generate coagulation, and exhibited much better RNAi potency and intracellular delivery compared with the conventional charge-complexed CPP/siRNA aggregates. Three different synthetic and purification methods were compared in order to optimize synthesis efficiency and product yield. The methodology using hetero-bifunctional NHS–PEG–OPSS as a crosslinker to synthesize LMWP–siRNA simplified the synthesis and purification process and produced the highest yield. These results pave the way towards siRNA biomimetic delivery and future clinical translation.

Self-assembly of peptide-based nanostructures： Synthesis and biological activity

Peptide-based nanostructures have received much attention in the field of drug targeting. In fact, peptides have many advantages such as simplicity of the structure, biocompatibility, and chemical diversity. Moreover, some peptides, which are called cell-penetrating peptides, can cross cellular membranes and carry small molecules, small interfering RNA, or viruses inside live cells. These molecules are often covalently or noncovalently linked to cargoes, thus forming amphiphilic conjugates that can self-assemble. Supramolecular nanostructures formed from peptides are used in nanomedicine as a carrier to protect a drug and to target cancer cells. This review explores aliphatic-chain-conjugated peptides and drug-conjugated peptides that can self-assemble. Special emphasis is placed on the synthesis procedure, nanostructure formation, and biological activity.

Development of a potent peptide inhibitor of estrogen receptor α

We have developed a facile N-terminus helix-nucleating strategy using an unnaturally tethered aspartic acid(TD strategy). Relatively weak nuclear translocation efficiency of TD PERM limits its further biological applications. A potent peptide inhibitor of estrogen receptor α(ER-α) with significantly increased cellular uptake and cellular distribution was developed by cell penetrating peptide attachment.The resulted peptide conjugate showed selective toxicity towards estrogen receptor positive cell lines and induced decreased transcription of estrogen receptor a downstream genes.

OCT4:A penetrant pluripotency inducer

Native OCT4 protein has the intrinsic ability of crossing cellular membranes to enter cells.This finding could revive efforts to induce pluripotency with proteins replacing nucleic acid-based approaches,and raises the intriguing question as to whether OCT4 can act non-cell-autonomously.

Fabrication of self-assembling nanofibers with optimal cell uptake and therapeutic delivery efficacy

Effective strategies to fabricate finite organic nanoparticles and understanding their structure-dependent cell interaction is highly important for the development of long circulating nanocarriers in cancer therapy.In this contribution,we will capitalize on our recent development of finite supramolecular nanofibers based on the self-assembly of modularly designed cationic multidomain peptides(MDPs)and use them as a model system to investigate structure-dependent cell penetrating activity.MDPs selfassembled into nanofibers with high density of cationic charges at the fiber-solvent interface to interact with the cell membrane.However,despite the multivalent charge presentation,not all fibers led to high levels of membrane activity and cellular uptake.The flexibility of the cationic charge domains on self-assembled nanofibers plays a key role in effective membrane perturbation.Nanofibers were found to sacrifice their dimension,thermodynamic and kinetic stability for a more flexible charge domain in order to achieve effective membrane interaction.The increased membrane activity led to improved cell uptake of membrane-impermeable chemotherapeutics through membrane pore formation.In vitro cytotoxicity study showed co-administering of water-soluble doxorubicin with membrane-active peptide nanofibers dramatically reduced the IC50 by eight folds compared to drug alone.Through these detailed structure and activity studies,the acquired knowledge will provide important guidelines for the design of a variety of supramolecular cell penetrating nanomaterials not limited to peptide assembly which can be used to probe various complex biological processes.