Low Molecular Weight Protamine Research Articles

Fine-tuning of liposome integrity for differentiated transcytosis and enhanced antitumor efficacy

Transcytosis-inducing nanomedicines have been developed to improve tumor extravasation. However, the fate during transcytosis across multicell layers and the structural integrity of the nanomedicines before reaching tumor cells could impact antitumor therapy. Here, a BAY 87–2243 (a hypoxia-inducible factor-1 inhibitor)-loaded liposomal system (HA-P-LBAY) modified by low molecular weight protamine (LMWP) and crosslinked by hyaluronic acid (HA) was constructed. This system could accomplish differentiate cellular transport in endothelial and tumor cells by fine-tuning its structural integrity, i.e. transcytosis across the endothelial cells while preserving structural integrity, facilitating subsequent retention and drug release within tumor cells via degradation-induced aggregation. In vitro cellular uptake and transwell studies demonstrated that HA-P-LBAY were internalized by endothelial cells (bEnd.3) via an active, caveolin and heparin sulfate proteoglycan (HSPG)-mediated endocytosis, and subsequently achieved transcytosis mainly through the ER/Golgi pathway. Moreover, the fluorescence resonance energy transfer (FRET) study showed that HA-crosslinking maintained higher integrity of HA-P-LBAY after transcytosis, more efficiently than electrostatic coating of HA (HA/P-LBAY). In addition, more HA-P-LBAY was retained in tumor cells (4T1) compared to HA/P-LBAY corresponding to its enhanced in vitro cytotoxicity. This may be attributed to better integrity of HA-P-LBAY post endothelial transcytosis and more degradation of HA in tumor cells, leading to more liposome aggregation and inhibition of their transcytosis, which was inferred by both TEM images and the HAase responsiveness assay proved by FRET. In vivo, HA-P-LBAY exhibited more potency in tumor suppression than the other formulations in both low and high permeability tumor models. This highlighted that fine-tuning of structural integrity of nanocarriers played a key role no matter whether the transcytosis of nanocarriers contributed to cellular transport. Collectively, this study provides a promising strategy for antitumor therapies by fine-tuning liposome integrity to achieve active trans-endothelial transport with structural integrity and selective aggregation for prolonged tumor retention.

Effect of Penetration Enhancer on the Structure of Stratum Corneum: On-Site Study by Confocal Polarized Raman Imaging.

Keratin and lipid structures in the stratum corneum (SC) are closely related to the SC barrier function. The application of penetration enhancers (PEs) disrupts the structure of SC, thereby promoting infiltration. To quantify these PE-induced structural changes in SC, we used confocal Raman imaging (CRI) and polarized Raman imaging (PRI) to explore the integrity and continuity of keratin and lipid structures in SC. The results showed that water is the safest PE and that oleic acid (OA), sodium dodecyl sulfate (SDS), and low molecular weight protamine (LMWP) disrupted the ordered structure of keratin, while azone and liposomes had less of an effect on keratin. Azone, OA, and SDS also led to significant changes in lipid structure, while LMWP and liposomes had less of an effect. Establishing this non-invasive and efficient strategy will provide new insights into transdermal drug delivery and skin health management.

Functional Poly(ε-caprolactone)/Poly(ethylene glycol) Copolymers with Complex Topologies for Doxorubicin Delivery to a Proteinase-Rich Tumor Environment

Doxorubicin (DOX)-loaded polymer nanoparticles based on poly(ethylene glycol)-poly(ε-caprolactone) copolymers with a complex macromolecular topology are proposed to tackle the matrix metalloproteinase (MMP)-rich tumor environment. Linear, 4-arm comb-like copolymers and 4-arm brush block copolymers were synthesized through a combination of ring opening polymerization and atom transfer radical polymerization, in order to control the molar mass distribution, the arm/brush architecture, as well as the final size and DOX loading of self-assembled nanoparticles obtained by nanoprecipitation. The optimized nanocarriers were conjugated with penetrating low molecular weight protamine peptides coupled to a polyanionic inhibitory domain cleavable by matrix metalloproteinase-2 (MMP2). DOX-loaded, MMP2-activable nanocarriers were evaluated in the context of glioblastoma (GBM), a brain tumor characterized by remarkable and relevant MMP2 expression. Uptake and cytotoxicity in patient-derived GBM cells correlated with the level of MMP2 enzymatic activity in a dose- and time-dependent manner. No effects were observed in nontumoral endothelial cells that do not express MMP2. Results demonstrated that, by tuning polymer topology and peptide sequence, nanoparticle self-assembly, DOX encapsulation, and delivery can be optimized for the development of an advanced treatment for MMP2-overexpressing tumors.

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14–22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Multifunctional nanoparticles recruiting hyaluronic acid ligand and polyplexes containing low molecular weight protamine and ATP-Sensitive DNA motif for doxorubicin delivery

Nanoparticles (NPs) that combine stimuli-triggered drug release and active targeting of tumors signify a dual smart strategy in chemotherapeutic inhibition of cancer growth. Recently, low molecular weight protamine (PrtLMW) has been utilized to replace its precursor protamine (Prt) in the clinic, as it exhibits less undesirable side effects. In this study, we compared the merits of using Prt and PrtLMW to assemble two NPs. Anticancer drug doxorubicin (Dox) was loaded into ATP-binding aptamer (AptATP), a DNA sequence that undergoes a conformational change when exposed to an Adenosine-5′-triphosphate (ATP) rich environment. Dox-AptATP then complexed with the positively charged Prt or PrtLMW, and the resulting cationic, condensed core was further coated with high molecular weight hyaluronic acid (HAHMW), a known ligand for tumor marker CD44. The resulting two NPs were characterized then inspected for selective uptake and release in CD44 overexpressing breast cancer cells (MDA-MB-231) using flow cytometry. The localization of the released Dox in the cytosol and nuclei of the cells was confirmed by confocal microscopy. A dose-dependent cytotoxic effect was observed by performing MTT assay. Annexin-V FITC apoptosis assay showed that the NPs were able to induce apoptosis efficiently. The replacement of Prt with PrtLMW proved to be beneficial in increasing the release rate of Dox, and it exhibited a substantially increased cytotoxic effect on the treated cancer cells.

Low molecular weight protamine-corticosteroid conjugate for topical treatment of psoriasis: A hypothesis

Psoriasis is a chronic, autoimmune inflammatory skin disease caused by disturbed keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis. In addition to long-term damages to skin, psoriasis also leads to numerous comorbidities, including psoriatic arthritis (PsA), cardiovascular and psychiatric complications, which seriously burdens the physical and psychological health of sufferers. Fluocinolone acetonide (FA) is a synthetic fluorine-containing potent corticosteroid, which can inhibit fibroblast proliferation, reduce skin inflammation and relieve itching. However, topical administration of FA for psoriasis treatment is restricted by its dose-dependent side effects mostly related to the high doses required or increased use frequency of FA owing to the chronic nature of psoriasis and the deprivations of FA. Cell penetration peptides (CPPs) based peptide–drug conjugates (PDCs) provide several benefits associated with delivery carriers, such as facilitating drugs' skin penetration, enhancing their cellular uptake, and improving their therapeutic efficacy. Low molecular weight protamine (LMWP) is an effective CPP from protamine enzymatic digestion. It holds an effective skin permeation capacity to act as a skin enhancer peptide. In this study, we hypothesize the construction of a reduction responsive LMWP-based FA conjugate nanostructure (LMWP-FA) for improving the therapeutic effects of FA in psoriasis. LMWP conjugation of FA would allow the enhanced skin permeation and promoted uptake by keratinocytes. The nanostructure and on-demand release under the intracellular reducing environment of LMWP-FA could locate FA in the psoriatic lesions to exert anti-inflammatory effects preventing its entry into the circulatory system. Taken together, the present hypothesis proposed a novel idea for FA to increase therapeutic efficiency and avoid side effects in the treatment of psoriasis.

Cell-Permeable Oct4 Gene Delivery Enhances Stem Cell-like Properties of Mouse Embryonic Fibroblasts.

Direct conversion of one cell type into another is a trans-differentiation process. Recent advances in fibroblast research revealed that epithelial cells can give rise to fibroblasts by epithelial-mesenchymal transition. Conversely, fibroblasts can also give rise to epithelia by undergoing a mesenchymal to epithelial transition. To elicit stem cell-like properties in fibroblasts, the Oct4 transcription factor acts as a master transcriptional regulator for reprogramming somatic cells. Notably, the production of gene complexes with cell-permeable peptides, such as low-molecular-weight protamine (LMWP), was proposed to induce reprogramming without cytotoxicity and genomic mutation. We designed a complex with non-cytotoxic LMWP to prevent the degradation of Oct4 and revealed that the positively charged cell-permeable LMWP helped condense the size of the Oct4-LMWP complexes (1:5 N:P ratio). When the Oct4-LMWP complex was delivered into mouse embryonic fibroblasts (MEFs), stemness-related gene expression increased while fibroblast intrinsic properties decreased. We believe that the Oct4-LMWP complex developed in this study can be used to reprogram terminally differentiated somatic cells or convert them into stem cell-like cells without risk of cell death, improving the stemness level and stability of existing direct conversion techniques.

Self-assembly of an aptamer-decorated chimeric peptide nanocarrier for targeted cancer gene delivery

In this study, we developed a peptide-based non-viral carrier decorated with aptamer to overcome the specific gene delivery barriers. The carrier (KLN/Apt) was designed to contain multiple functional segments, including 1) two tandem repeating units of low molecular weight protamine (LMWP) to condense DNA into stable nanosize particles and protect it from enzymatic digestion, 2) AS1411 aptamer as targeting moiety to target nucleolin and promote carrier internalization, 3) a synthetic pH-sensitive fusogenic peptide (KALA) for disrupting endosomal membranes and enhancing cytosol escape of the nanoparticles, and 4) a nuclear localization signal (NLS) for active cytoplasmic trafficking and nuclear delivery of DNA. The obtained results revealed the developed carrier capacity in terms of specific cell targeting, overcoming cellular gene delivery barriers, and mediating efficient gene transfection. The KLN/pDNA/aptamer nanoparticles offer remarkable potential for the conceptual design and formation of promising multi-functionalized carriers towards the most demanding therapeutic applications.

Evaluation of the efficiency of modified PAMAM dendrimer with low molecular weight protamine peptide to deliver IL-12 plasmid into stem cells as cancer therapy vehicles.

Interleukin 12 (IL-12) is considered as an important molecule for cancer immunotherapy with significant roles in hindering tumor activity, mostly mediated by tumor-associated macrophages and anti-angiogenic factors. Mesenchymal stem cells (MSCs) have been come out as promising carriers to increase the accumulation of drug/gene in tumor sites. As a vehicle, MSCs have various advantages, including tumor-specific propensity and migratory ability; however, they have limited transfection efficiency, compared to other cells. In this study, we introduced a novel delivery system based on poly-(amidoamine) (PAMAM) (G5) to deliver a plasmid encoding IL-12 to MSCs. Initially, 30% of the amine surface of PAMAM was substituted by 10-bromodecanoic acid. Then, the low molecular weight of protamine peptide was conjugated to PAMAM and PAMAM-alkyl with N-succinimidyl 3-(2-pyridyldithio) propionate as a linker. Physicochemical properties of this modified PAMAM were evaluated, including size and surface charge, toxicity, transfection efficiency to deliver reporter and IL-12 genes into MSCs and finally the migration potential of the engineered stem cells into cancer and normal cell lines (HepG2 and NIH/3T3). The results showed that alkyl-peptide modified PAMAM with low toxicity had a higher potential to deliver green fluorescent protein and IL-12 genes to stem cells, than PMAMAM, PAMAM-alkyl and PAMAM-peptide. These engineered stem cells had a greater ability to migrate to cancer cells than normal cells. It can be concluded that engineered stem cells containing the IL-12 gene can be considered as an efficient cell carrier for cancer immunotherapy. Further clinical studies are needed to confirm these results.

Erythrocytes internalize nanoparticles functionalized with low molecular weight protamine

Erythrocytes have been extensively investigated as drug delivery vehicles. However, many existing methods for loading therapeutic biomolecules into erythrocytes perturb the cell membrane, resulting in a loss of erythrocyte structural integrity and recognition by the host immune system. To overcome this challenge, we developed a novel nanoparticle carrier for transporting proteins into intact erythrocytes. We designed and characterized a nanoparticle where a poly-L-lysine-grafted-poly(ethylene) glycol copolymer was used to encapsulate a model protein, bovine serum albumin. The nanoparticle was functionalized with either an erythrocyte targeting peptide ERY1, the cell-penetrating peptide low molecular weight protamine, or a combination of the two peptides. The three resulting constructs, ENP, LNP, and ELNP, had a core/shell structure and, depending on functionalization, were approximately 30–90 nm in diameter and had negative surface charge. Esterase-like activity of the encapsulated protein was not affected by encapsulation. Under physiological conditions, nanoparticles caused no significant hemolysis and were resistant to dissociation by surfactants. The three nanoparticle constructs were evaluated using confocal microscopy and flow cytometry for their ability to bind and internalize within intact erythrocytes. LNPs showed significant binding and internalization into intact erythrocytes, with ~90% of erythrocytes containing particles after 24 h of co-incubation. Internalization occurred without adversely affecting erythrocyte membrane structure. Together, these results demonstrate that low molecular weight protamine–functionalized nanoparticles may serve as a universal platform to deliver protein therapeutics into erythrocytes.

Multifunctional peptides based on low molecular weight protamine (LMWP) in the structure of polyplexes and lipopolyplexes: Design, preparation and gene delivery characterization

The development of an effective and safe gene carrier is one of the main challenges in gene therapy. In this study, we designed two new cationic peptides (VV45 and VV32) based on low molecular weight protamine (LMWP) which were upgraded by adding a histidine tag (H10) and nuclear localization signal (NLS) peptide and evaluated their transfection efficiency and cytotoxicity in the formulation of Peptide-DNA (PD) and Liposome-Peptide-DNA (LPD) complexes. Using the gel retardation assay for assessment of condensation ability, three carriers to plasmid (C/P) ratios of 1, 3, and 6 for PD and LPD formulations were selected, and prepared vectors were evaluated in terms of size distribution and zeta potential. All vectors in deionized water (DW) had a size below 200 nm (PD complexes: 65–120 nm, and LPD complexes: 140–185 nm) and a positive zeta potential (PD: 3–15 mV, and LPD: 32–48 mV). Generally, the LPD complexes revealed higher GFP expression than PD complexes, up to four-folds. Compared to,polyethyleneimine 25 kDa, as a transfection standard, the LPD VV45 at C/P ratios of 3, 6 and LPD VV32 at C/P ratio of 6 had a higher transfection. In terms of cytotoxicity, PD complexes have no cytotoxicity, and at the LPD complexes, increasing in C/P ratio decreased the cell viability to 30–50%. So newly designed peptides are effective gene carriers in the formulation of LPD with a promising safety profile in polyplex formulation.

DARPin Ec1-LMWP protein scaffold in targeted delivery of siRNA molecules through EpCAM cancer stem cell marker.

This study is to investigate the binding ability of Designed Ankyrin Repeat Proteins type Ec1that was fused to Low Molecular Weight Protamine (DARPin Ec1-LMWP) protein scaffold to the Epithelial Cell Adhesion Molecule (EpCAM) Cancer Stem Cell (CSC) marker and its efficiency in targeted delivery of small interfering RNA (siRNA) molecules into the studied cells. Gene fragment encoding the DARPIn Ec1-LMWP fusion protein was subcloned into pET28a expression vector following molecular docking studies performed to examine the affinity of the fusion protein towards EpCAM marker. The binding of the siRNA to the expressed fusion protein was tested through native PAGE. The toxicity of the fusion protein was tested by MTT assay. Attachment of the complex to the EpCAM marker was investigated by flow cytometry and delivery of siRNA into the cells was assessed by fluorescence microscopy. The expressed 21.6kDa DARPin Ec1-LMWP fusion protein was purified and showed no cytotoxicity on tested cells. Arginine rich LMWP was efficiently bounded to the negatively charged siRNA molecule. Successful attachment of the fusion protein:siRNA complex to the EpCAM marker and its internalization into MCF-7 breast cancer cell line were confirmed. Here for the first time the recombinant DARPin Ec1-LMWP protein scaffold was designed and tested for targeting EpCAM surface marker and successful internalization of the siRNA into MCF-7 cells.

A self-assembly and stimuli-responsive fusion gelonin delivery system for tumor treatment

Ribosome-inactivating proteins (RIPs) are potent protein toxins for cancer therapy, and they have strong ability to inhibit protein synthesis and induce cell death via inactivation of ribosomes in eukaryotic cells. However, the delivery of RIPs has been a challenging task due to their large molecular weight and lack of targeting property. Low molecular weight protamine (LMWP), a transmembrane peptide, has been proved to effectively promote transmembrane transportation, whereas the enzyme-activatable system can enhance the specificity by enhancing the tumor drug concentration through enzymatic reaction. We herein constructed a self-assembly and stimuli-responsive fusion gelonin delivery system. Gelonin, a typical RIP protein, was assembled with nickel ferrite nanoparticles by self-assembling between hexa-histidine tag (His-tagged) and nickel ions. Both in vitro and in vivo results indicated that the magnetic nanoparticle carriers and the applied linkers did not damage the pharmaceutical effect of gelonin, and the whole drug delivery system showed good biocompatibility, sensitive selectivity, and significantly enhanced cytotoxic activity. This in turn presented theranostic nanoparticles as efficient delivery vehicle for clinical use.

Design, Synthesis, and In Vitro Evaluation of Low Molecular Weight Protamine (LMWP)-Based Amphiphilic Conjugates as Gene Delivery Carriers.

Development of efficient non-viral carriers is one of the major challenges of gene delivery. In the current study, we designed, synthesized, and evaluated the in vitro gene delivery efficiency of novel amphiphilic constructs composed of cholesterol and low molecular weight protamine (LMWP: VSRRRRRRGGRRRR) peptide. Vectors having both hydrophobic and hydrophilic moieties were evaluated in terms of particle size and charge, DNA condensation ability, cytotoxicity, and gene transfection efficiency. The prepared vectors spontaneity self-assembled into the liposome-like particles with a high local positive density. The nano-vehicle A (H5-LMWP-Cholestrol) and nano-vehicle B (LMWP-Cholesterol) could form micelles at concentrations above 50μg/mL and 65μg/mL, respectively. The gel retardation assay showed that nano-vehicles A and B could condense pDNA more efficiently than the corresponding unconjugated peptides. The mean of size and zeta potential of complexed nano-vehicle A at N/P ratios of 5, 15, and 30 were 151nm and 23mv, and those of nano-vehicle B were 224nm and 19mv, respectively. In terms of transfection efficiency, the designed nano-vehicles showed almost two-fold higher gene expression level compared to PEI 25kDa at optimal N/P ratios, and also exhibited negligible cytotoxicity on a model cancer cell, Neuro 2a. The findings of the present study revealed that these cationic micelles can be promising candidates as non-viral gene delivery vehicles.

Oral delivery system for low molecular weight protamine-dextran-poly(lactic-co-glycolic acid) carrying exenatide to overcome the mucus barrier and improve intestinal targeting efficiency.

Aim: This study aimed to explore the effect of nanoparticles loaded with exenatide in overcoming the mucus barrier and improving intestinal targeting efficiency, to improve the oral bioavailability. Materials & methods: Low molecular weight protamine (LMWP)-dextran-poly(lactic-co-glycolic acid) was used to create LMWP-dextran-poly(lactic-co-glycolic acid)-nanoparticles (LDPs) encapsulating exenatide-Zn2+ complex.Results & conclusion: LDPs showed improved penetration of the mucus barrier, and LMWP was helpful for mediating cell translocation through protein transduction domains. The absorption sites and distribution rates of LDPs were verified by intestinal localization experiments and in vivo distribution experiments. Cell uptake and transmembrane experiments confirmed the absorption efficiency in the intestinal epithelium. Furthermore, the relative bioavailability after oral administration of exenatide-Zn2+-LDPs was 8.4%, with a significant hypoglycemic effect on Type 2 diabetic mice.

Simple in Vivo Gene Editing via Direct Self-Assembly of Cas9 Ribonucleoprotein Complexes for Cancer Treatment.

Cas9 ribonucleoprotein (RNP)-mediated delivery has emerged as an ideal approach for in vivo applications. However, the delivery of Cas9 RNPs requires electroporation or lipid- or cationic-reagent-mediated transfection. Here, we developed a carrier-free Cas9 RNP delivery system for robust gene editing in vivo. For simultaneous delivery of Cas9 and a guide RNA into target cells without the aid of any transfection reagents, we established a multifunctional Cas9 fusion protein (Cas9-LMWP) that forms a ternary complex with synthetic crRNA:tracrRNA hybrids in a simple procedure. Cas9-LMWP carrying both a nuclear localization sequence and a low-molecular-weight protamine (LMWP) enables the direct self-assembly of a Cas9:crRNA:tracrRNA ternary complex (a ternary Cas9 RNP) and allows for the delivery of the ternary Cas9 RNPs into the recipient cells, owing to its intrinsic cellular and nuclear translocation ability with low immunogenicity. To demonstrate the potential of this system, we showed extensive synergistic anti-KRAS therapy (CI value: 0.34) via in vitro and in vivo editing of the KRAS gene by the direct delivery of multifunctional Cas9 RNPs in lung cancer. Thus, our carrier-free Cas9 RNP delivery system could be an innovative platform that might serve as an alternative to conventional transfection reagents for simple gene editing and high-throughput genetic screening.

The use of low molecular weight protamine to enhance oral absorption of exenatide

Although oral delivery of exenatide has significant advantages, its poor permeability through intestinal epithelial membranes and rapid digestion by pepsin and ereptase in the gastrointestinal tract make effective oral delivery of exenatide a formidable challenge. In this study, we constructed a zinc ion (Zn2+) and exenatide complex functionalized nanoparticle (NP) oral delivery system to overcome the above-mentioned issue. Polyethylene glycol-poly(lactic-co-glycolic acid) (PEG-PLGA) was used as a drug carrier to escape enzymatic degradation in the gastrointestinal tract, and low molecular weight protamine (LMWP) was used as a functional group to increase penetration of NPs into the intestinal epithelium. The functionalized NPs exhibited significantly improved penetration across the intestinal epithelium, as shown by cell uptake and transmembrane transport experiments. Moreover, a significant hypoglycemic effect was observed in diabetic rats. The relative bioavailability of the orally administered functionalized NPs vs. subcutaneous injection was 7.44%, 29-fold that of the exenatide-Zn2+ solution group. These findings indicate that our modification could effectively improve exenatide treatment.

Construction and Characterization of Cell-Penetrating Peptide-Fused Fibroblast Growth Factor and Vascular Endothelial Growth Factor for an Enhanced Percutaneous Delivery System.

The exogenous administration of growth factors has been examined for treating wounds and such factors serve as cosmetic agents for skin regeneration. However, the topical application of growth factors is hampered by their limited percutaneous absorption. In this study, we genetically modified basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) using a cell-penetrating peptide to facilitate their permeation into skin and to avoid multiple steps in the chemical or physical modification of biomaterials. Low-molecular-weight protamine (LMWP)-fused bFGF and VEGF-A (LMWP-bFGF and LMWP-VEGF-A) were designed by serial polymerase chain reaction (PCR)-mediated addition of the LMWP codons onto the bFGF and VEGF-A genes and then produced in Escherichia coli. The purified LMWP-bFGF and LMWP-VEGF-A represented >90% of the total proteins, as determined by SDS-PAGE and densitometric quantification, and their actual molecular weights, determined by mass spectroscopy, were 19,026 and 15,715 Da, respectively, corresponding to the theoretical values. N-terminal amino acid sequencing analyses confirmed the N-terminal conjugation of LMWP to bFGF and VEGF-A. Immunoblotting analyses using antibodies against bFGF and VEGF-A, together with the proliferative effects of LMWP-bFGF and LMWP- VEGF-A on human keratinocytes and fibroblasts, demonstrated that the original biological activity of each growth factor was not altered by LMWP conjugation. In addition, the LMWP conjugation did not induce further cytotoxic effects on the skin cells, while the cell membrane-penetrating activities of LMWP-bFGF and LMWP-VEGF-A were significantly enhanced compared with the respective unconjugated growth factors. These results suggest that LMWP-bFGF and LMWP-VEGF-A can be used as effective topical therapeutic or cosmetic agents for skin regeneration and anti-aging treatments.

Preparation and In Vitro Evaluation of Elastic Nanoliposomes for Topical Delivery of Highly Skin-Permeable Growth Factors.

Percutaneous delivery of growth factors is often used to treat wounds, and for cosmetic purposes, as a way of accelerating healing and skin regeneration, respectively. However, the therapeutic effects of growth factors are diminished by their poor absorption when delivered percutaneously, in addition to their rapid degradation by proteinases. To overcome these obstacles, we constructed two skin-permeable compounds. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) were both genetically paired with low-molecular-weight protamine (LMWP), to yield the compounds LMWP-bFGF and LMWP-VEGF-A, respectively. The molecular weights and N-terminal amino acid sequences of LMWP-bFGF and LMWP-VEGF-A confirmed that the N-terminus-specific conjugation of LMWP with bFGF and VEGF-A had been successful. The biological abilities of the native factors to stimulate human fibroblast (CCD-986sk) and endothelial cell proliferation were preserved. Both compounds significantly promoted wound (scratch) recovery and enhanced procollagen type I C-peptide synthesis in CCD-986sk cells (to levels 184 and 133% those of the native compounds, respectively). The LMWP-conjugated growth factors were significantly more permeable than the native forms (by 7.29- and 29.22-fold, respectively). Finally, encapsulation of the compounds in positively charged elastic nanoliposomes (115 ± 1.54 nm in diameter with a zeta potential of 57.2 ± 3.05 mV) further improved both permeation and stability. Thus, nanoliposomes loaded with LMWP-conjugated growth factors are expected to enhance skin regeneration; the materials will find applications in wound-healing therapies and anti-wrinkle cosmetics.

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.