Efficacy of Alum-Adjuvanted Peptide and Carbohydrate Conjugate Vaccine Candidates against Group A Streptococcus Pharyngeal Infection in a Non-Human Primate Model.

Vaccine development against group A Streptococcus (GAS) has gained traction in the last decade, fuelled by recognition of the significant worldwide burden of the disease. Several vaccine candidates are currently being evaluated in preclinical and early clinical studies. Here, we investigate two conjugate vaccine candidates that have shown promise in mouse models of infection. Two antigens, the J8 peptide from the conserved C-terminal end of the M protein, and the group A carbohydrate lacking N-acetylglucosamine side chain (ΔGAC) were each conjugated to arginine deiminase (ADI), an anchorless surface protein from GAS. Both conjugate vaccine candidates combined with alum adjuvant were tested in a non-human primate (NHP) model of pharyngeal infection. High antibody titres were detected against J8 and ADI antigens, while high background antibody titres in NHP sera hindered accurate quantification of ΔGAC-specific antibodies. The severity of pharyngitis and tonsillitis signs, as well as the level of GAS colonisation, showed no significant differences in NHPs immunised with either conjugate vaccine candidate compared to NHPs in the negative control group.

Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing.

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.

Formulation and Biological Evaluation of Mesoporous Silica Nanoparticles Loaded with Combinations of Sortase A Inhibitors and Antimicrobial Peptides.

This study aimed to develop synergistic therapies to treat superbug infections through the encapsulation of sortase A inhibitors (SrtAIs; trans-chalcone (TC), curcumin (CUR), quercetin (QC), or berberine chloride (BR)) into MCM-41 mesoporous silica nanoparticles (MSNs) or a phosphonate-modified analogue (MCM-41-PO3-) to overcome their poor aqueous solubility. A resazurin-modified minimum inhibitory concentration (MIC) and checkerboard assays, to measure SrtAI synergy in combination with leading antimicrobial peptides (AMPs; pexiganan (PEX), indolicidin (INDO), and [I5, R8] mastoparan (MASTO)), were determined against methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The results demonstrated that the MCM-41 and MCM-41-PO3- formulations significantly improved the aqueous solubility of each SrtAI. The MICs for SrtAI/MCM-41-PO3- formulations were lower compared to the SrtAI/MCM-41 formulations against tested bacterial strains, except for the cases of BR/MCM-41 and QC/MCM-41 against P. aeruginosa. Furthermore, the following combinations demonstrated synergy: PEX with TC/MCM-41 (against all strains) or TC/MCM-41-PO3- (against all strains except P. aeruginosa); PEX with BR/MCM-41 or BR/MCM-41-PO3- (against MSSA and MRSA); INDO with QC/MCM-41 or QC/MCM-41-PO3- (against MRSA); and MASTO with CUR/MCM-41 (against E. coli). These combinations also reduced each components' toxicity against human embryonic kidney cells. In conclusion, MCM-41 MSNs provide a platform to enhance SrtAI solubility and demonstrated antimicrobial synergy with AMPs and reduced toxicity, providing novel superbug treatment opportunities.

Developing GLP-1 Conjugated Self-Assembling Nanofibers Using Copper-Catalyzed Alkyne-Azide Cycloaddition and Evaluation of Their Biological Activity.

Glucagon-like peptide-1 GLP-1 is a gut-derived peptide secreted from pancreatic ß-cells that reduces blood glucose levels and body weight; however, native GLP-1 (GLP-1(7-36)-NH2 and GLP-1(7-37)) have short in vivo circulation half-lives (∼2 min) due to proteolytic degradation and rapid renal clearance due to its low molecular weight (MW; 3297.7 Da). This study aimed to improve the proteolytic stability and delivery properties of glucagon-like peptide-1 (GLP-1) through modifications that form nanostructures. For this purpose, N- (NtG) and C-terminal (CtG), and Lys26 side chain (K26G) alkyne-modified GLP-1 analogues were conjugated to an azide-modified lipidic peptide (L) to give N-L, C-L, and K-26-L, respectively; or CtG was conjugated with a fibrilizing self-assembling peptide (SAP) (AEAEAKAK)3 to yield C-S, using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). N-L demonstrated the best serum stability (t1/2 > 48 h) compared to K-26-L (44 h), C-L (20 h), C-S (27 h), and the parental GLP-1(7-36;A8G)-NH2 (A8G) (19 h) peptides. Each conjugate demonstrated subnanomolar hGLP-1RA potency, and none demonstrated toxicity toward PC-3 cells at concentrations up to 1 µM. Each analogue was observed by transmission electron microscopy to form fibrils in solution. K-26-L demonstrated among the best human serum stability (t1/2 = 44 h) and similar hGLP-1RA potency (EC50 48 pM) to C-S. In conclusion, this study provided an alternative to lipid modification, i.e., fibrillizing peptides, that could improve pharmacokinetic parameters of GLP-1.

Neutralisation of adeno-associated virus transduction by human vitreous humour.

Neutralising antibodies (NAbs), caused by past adeno-associated virus (AAV) infection, represent a critical challenge for AAV-mediated gene therapy, with even low NAb titres capable of inhibiting gene transfer, however in protein-rich environments such as the vitreous it is expected that other constituents could also interact with the transduction process. Inhibition of AAV2/2, AAV2/5, AAV2/6 and AAV2/8 transduction by human vitreous humour (VH) obtained from 80 post-mortem eye cups was investigated in this report, with clinically relevant vitreous dilutions as low as 1:2. Unexpectedly, the highest prevalence of inhibition of transduction was observed against AAV2/6, with 66% of tested samples displaying neutralisation at a 1:2 VH dilution. Only two samples showed inhibition of AAV2/8, indicating this serotype is an attractive vector for use in non-vitrectomised eyes of unscreened individuals. Levels of anti-AAV NAbs observed in the VH were much lower than previously observed in serum of a similar Australian population. Among ten tested eye cup pairs, we observed only small variation in anti-AAV NAbs levels between the left and right eye cups. Interaction with 1:2 diluted VH had an augmentation effect on AAV2/8 transduction (p = 0.004), a phenomenon which was not due to albumin or transferrin and which, if developed, might benefit the use of AAV2/8 in clinical settings.

Supercritical fluid assembly of albendazole liposomes targeting gastrin-releasing peptide receptor overexpressing tumors.

Aim: To develop albendazole (ABZ)-loaded bombesin(6-14) (BBN(6-14)) functionalized liposomes for targeting GRPR to enhance delivery to cancer cells. Materials & methods: ABZ-loaded liposomes were formulated using supercritical CO2 technology; functionalized with a GRPR-targeted lipid-anchored BBN(6-14) peptide; and evaluated for effects on cell viability, particle size and targeted cell uptake. Results: BBN(6-14)-coated ABZ liposomes decreased cell viability compared with nonfunctionalized ABZ liposomes. The level of GRPR expression positively correlated with intracellular uptake and decreased cell viability. The reduced cell viability, higher cell uptake and GRPR expression were observed in the order PC-3 > Caco-2 > HepG2 cells. Conclusion: BBN(6-14)-functionalized ABZ liposomes showed enhanced reduction in cell viability compared with nonfunctionalized ABZ liposomes.

Semisynthetic, self-adjuvanting vaccine development: Efficient, site-specific sortase A-mediated conjugation of Toll-like receptor 2 ligand FSL-1 to recombinant protein antigens under native conditions and application to a model group A streptococcal vaccine.

Protein antigens are, in general, weakly immunogenic, and therefore require co-delivery with adjuvants to stimulate potent immune responses. The fusion of (poly)peptide antigens to immunostimulatory adjuvants (e.g. Toll-like receptor (TLR) agonists) has been demonstrated to greatly improve vaccine potency compared to mixtures of antigen and adjuvant. Chemical approaches, to enable the rapid, site-specific and high-yielding linkage of TLR2 ligands to recombinant protein antigens, have been previously optimized. These approaches require the use of denaturing conditions to ensure high reaction yields, which limits their application, as maintenance of native protein folding is necessary to elicit antibodies against conformational epitopes. Here, this work aimed to optimize an alternative method, to ensure the efficient bioconjugation of TLR2 ligands onto folded protein antigens. An enzyme-mediated approach, using Staphylococcus aureus sortase A (or a penta mutant with enhanced efficiency), was optimized for reaction yield and time, as well as enzyme type and amount. This approach enabled the site-specific conjugation of the TLR2-agonist fibroblast-stimulating lipopeptide-1 (FSL-1) onto a model group A Streptococcus (GAS) recombinant polytope antigen under conditions that maintain protein folding, yielding a homogeneous, molecularly-defined product, with ligation yields as high as 90%. Following intramuscular (IM) administration of the ligation product to humanized plasminogen AlbPLG1 mice, high-titer, antigen-specific IgG antibodies were observed, which conferred protection against subcutaneous challenge with GAS strain 5448. In comparison, mixtures of the GAS antigen with aluminum hydroxide or FSL-1 failed to provide protection, with the FSL-1 mixture yielding ~1000-fold lower antigen-specific IgG antibody titers, and the mixture with alum yielding a Th2-biased response compared to the more balanced Th1/Th2 responses observed with the FSL-1 conjugate. Overall, a FSL-1 bioconjugation method for the efficient production of antigen-TLR2 agonist conjugates, which maintain protein folding, was produced, with broad utility for the development of self-adjuvanting vaccines against subunit protein antigens.

Peptide-based targeted polymeric nanoparticles for siRNA delivery.

The development of polymer-based nanoparticulate delivery systems for siRNA is important for the clinical success of gene therapy. However, there are some major drawbacks that need to be overcome. Short interfering RNA (siRNA) has been investigated as a potential therapeutic drug to silence disease-associated genes, but its usage is limited due to the lack of effective and safe nanocarriers. In this study, DOPE-PEI, a nanoparticle consisting of the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) conjugated with low-molecular-weight, 600 Da, branched polyethylenimine (PEI) was produced and optimized for siRNA delivery. This delivery system was modified with other components such as 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)2000] (DOPE-PEG2K), DOPE-PEG3.4K-bombesin and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine/1,2-dioleoyl-3-trimethylammonium-propane (DOPE/DOTAP) and tested on PC-3 cells. The conjugation of DOPE to PEI polymer (DOPE-PEI) improved the efficiency of PEI to deliver siRNA into the cytosol and knockdown genes, but demonstrated high toxicity. The addition of DOPE-PEG2K reduced cellular toxicity by masking the surface positive charge of the DOPE-PEI/siRNA complex, with the incorporation of a gastrin-releasing peptide receptor (GRPR) targeting peptide and DOPE/DOTAP components improving the cellular uptake of siRNA into targeted cells and the siRNA knockdown efficiency.

Soil bacterial diffusible and volatile organic compounds inhibit Phytophthora capsici and promote plant growth.

Biotic interactions through diffusible and volatile organic compounds (VOCs) are frequent in nature. Soil bacteria are well-known producers of a wide range of volatile compounds (both organic and inorganic) with various biologically relevant activities. Since the last decade, they have been identified as natural biocontrol agents. Volatiles are airborne chemicals, which when released by bacteria, can trigger plant responses such as defence and growth promotion. In this study, we tested whether diffusible and volatile organic compounds (VOCs) produced by soil bacterial isolates exert anti-oomycete and plant growth-promoting effects. We also investigated the effects of inoculation with VOC-producing bacteria on the growth and development of Capsicum annuum and Arabidopsis thaliana seedlings. Our results demonstrate that organic VOCs emitted by bacterial antagonists negatively influence mycelial growth of the soil-borne phytopathogenic oomycete Phytophthora capsici by 35% in vitro. The bacteria showed plant growth promoting effects by stimulating biomass production, primary root growth and root hair development. Additionally, we provide evidence to suggest that these activities were deployed by the emission of either diffusible organic compounds or VOCs. Bacterial VOC profiles were obtained through solid phase microextraction (SPME) and analysis by gas chromatography coupled with mass spectrometry (GC-MS). This elucidated the main volatiles emitted by the isolates, which covered a wide range of aldehydes, alcohols, esters, carboxylic acids, and ketones. Collectively, twenty-five VOCs were identified to be produced by three bacteria; some being species-specific. Our data show that bacterial volatiles inhibits P. capsici in vitro and modulate both plant growth promotion and root system development. These results confirm the significance of soil bacteria and highlights that ways of harnessing them to improve plant growth, and as a biocontrol agent for soil-borne oomycetes through their volatile emissions deserve further investigation.

Glucagon-Like Peptide-1 Receptor Agonists and Strategies To Improve Their Efficiency.

Type 2 diabetes mellitus (T2DM) is increasing in global prevalence and is associated with serious health problems (e.g., cardiovascular disease). Various treatment options are available for T2DM, including the incretin hormone glucagon-like peptide-1 (GLP-1). GLP-1 is a therapeutic peptide secreted from the intestines following food intake, which stimulates the secretion of insulin from the pancreas. The native GLP-1 has a very short plasma half-life, owning to renal clearance and degradation by the enzyme dipeptidyl peptidase-4. To overcome this issue, various GLP-1 agonists with increased resistance to proteolytic degradation and reduced renal clearance have been developed, with several currently marketed. Strategies, such as controlled release delivery systems, methods to reduce renal clearance (e.g., PEGylation and conjugation to antibodies), and methods to improve proteolytic stability (e.g., stapling, cyclization, and glycosylation) provide means to further improve the ability of GLP-1 analogs. These will be discussed in this literature review.

Gastrin-releasing peptide receptor-targeted hybrid peptide/phospholipid pDNA/siRNA delivery systems.

Aim: To develop a peptide/phospholipid hybrid system for gastrin-releasing peptide receptor (GRPR)-targeted delivery of pDNA or siRNA. Materials & methods: A multifunctional GRPR-targeted peptide R9-K(GALA)-BBN(6-14) was combined with a phospholipid oligonucleotide delivery system (1:1 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 1,2-dioleoyl-3-trimethylammonium-propane) and evaluated for pDNA and siRNA delivery in terms of complex size, toxicity, receptor-targeted delivery and gene expression or knockdown efficiency. Results: By combining peptide and phospholipid delivery systems, synergistic improvements in gene expression and knockdown were observed when compared with either system alone. The optimized formulation demonstrated high levels of EGFP expression and EGFP knockdown, GRPR-targeted delivery, enhanced endosomal release and minimal toxicity. Conclusion: The peptide/phospholipid hybrid system provides efficient GRPR-targeted DNA/siRNA delivery.

An Experimental Group A Streptococcus Vaccine That Reduces Pharyngitis and Tonsillitis in a Nonhuman Primate Model.

Group A Streptococcus (GAS) infections account for an estimated 500,000 deaths every year. This bacterial pathogen is responsible for a variety of mild and life-threatening infections and the triggering of chronic autoimmune sequelae. Pharyngitis caused by group A Streptococcus (GAS), but not asymptomatic GAS carriage, is a prerequisite for acute rheumatic fever (ARF). Repeated bouts of ARF may trigger rheumatic heart disease (RHD), a major cause of heart failure and stroke accounting for 275,000 deaths annually. A vaccine that prevents pharyngitis would markedly reduce morbidity and mortality from ARF and RHD. Nonhuman primates (NHPs) have been utilized to model GAS diseases, and experimentally infected rhesus macaques develop pharyngitis. Here we use an NHP model of GAS pharyngitis to evaluate the efficacy of an experimental vaccine, Combo5 (arginine deiminase [ADI], C5a peptidase [SCPA], streptolysin O [SLO], interleukin-8 [IL-8] protease [SpyCEP], and trigger factor [TF]), specifically designed to exclude GAS components potentially linked to autoimmune complications. Antibody responses against all Combo5 antigens were detected in NHP serum, and immunized NHPs showed a reduction in pharyngitis and tonsillitis compared to controls. Our work establishes the NHP model as a gold standard for the assessment of GAS vaccines.IMPORTANCE GAS-related diseases disproportionally affect disadvantaged populations (e.g., indigenous populations), and development of a vaccine has been neglected. A recent strong advocacy campaign driven by the World Health Organization and the International Vaccine Institute has highlighted the urgent need for a GAS vaccine. One significant obstacle in GAS vaccine development is the lack of a widely used animal model to assess vaccine efficacy. Researchers in the field use a wide range of murine models of infection and in vitro assays, sometimes yielding conflicting results. Here we present the nonhuman primate pharyngeal infection model as a tool to assess vaccine-induced protection against colonization and clinical symptoms of pharyngitis and tonsillitis. We have tested the efficacy of an experimental vaccine candidate with promising results. We believe that the utilization of this valuable tool by the GAS vaccine research community could significantly accelerate the realization of a safe and effective GAS vaccine for humans.

Advances in Targeted Gene Delivery.

Gene therapy has the potential to treat both acquired and inherited genetic diseases. Generally, two types of gene delivery vectors are used - viral vectors and non-viral vectors. Non-viral gene delivery systems have attracted significant interest (e.g. 115 gene therapies approved for clinical trials in 2018; clinicaltrials.gov) due to their lower toxicity, lack of immunogenicity and ease of production compared to viral vectors. To achieve the goal of maximal therapeutic efficacy with minimal adverse effects, the cell-specific targeting of non-viral gene delivery systems has attracted research interest. Targeting through cell surface receptors; the enhanced permeability and retention effect, or pH differences are potential means to target genes to specific organs, tissues, or cells. As for targeting moieties, receptorspecific ligand peptides, antibodies, aptamers and affibodies have been incorporated into synthetic nonviral gene delivery vectors to fulfill the requirement of active targeting. This review provides an overview of different potential targets and targeting moieties to target specific gene delivery systems.

Dispersibility of phospholipids and their optimization for the efficient production of liposomes using supercritical fluid technology.

Liposomes are promising delivery vehicles and offer the added drawcard of being able to be made functional to target tissues such as cardiac muscle and cancerous cells. Current methods to manufacture liposomes need to be improved and supercritical fluid (SCF) technologies may offer a solution. Herein, the dispersibility of six different phospholipids (PLs) was determined in supercritical carbon dioxide (scCO2). 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) showed the highest post-processing dispersibility, while 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) showed no dispersibility in scCO2 at the assessed experimental conditions. The zetasizer results showed that the SCF conditions at 37 °C, 250 bar and 200 RPM for 60 min provided nanoparticles with the narrowest polydispersity index (PDI) and a spherical shape as shown by cryo-transmission electron microscopy (Cryo-TEM). The mean diameter of liposomes using the SCF method for DSPC-PEGylated and DOPC-PEGylated liposomes was 98.3 ±â€¯3.3 nm and 124.5 ±â€¯4.1 nm, while using the thin film method it was 153.6 ±â€¯4.5 nm and 131.3 ±â€¯3.4 nm, respectively. A size-based stability evaluation of the scCO2-prepared liposomes stored at different temperatures (25 °C, 4 °C and -20 °C) was compared to that of the thin film method over a period of 3 months. The current study provides a possible green alternative SCF method to preparing liposomes that is less laborious, time saving, and a low energy process.

Glucagon-Like Peptide-1 (GLP-1)-Based Therapeutics: Current Status and Future Opportunities beyond Type†2 Diabetes.

Glucagon-like peptide-1 (GLP-1) is secreted by intestinal L-cells following food intake, and plays an important role in glucose homeostasis due to its stimulation of glucose-dependent insulin secretion. Further, GLP-1 is also associated with protective effects on pancreatic ß-cells and the cardiovascular system, decreased appetite, and weight loss, making GLP-1 derivatives an exciting treatment for type 2 diabetes and obesity. Despite these benefits, wild-type GLP-1 exhibits a short circulation time due to its poor metabolic stability and rapid renal clearance, and must be administered by injection, making it a poor therapeutic agent. Many strategies have been used to improve the circulation time of GLP-1 (e.g., mutations, unnatural amino acids, depot formulations, use of exendin-4 sequences, and fusions with high-molecular-weight proteins or polymers), with its therapeutic utility further improved by adding agonist activity for gastric inhibitory peptide and glucagon receptors. This minireview focuses on strategies that have been used to improve the pharmacokinetics of GLP-1 and provides an overview of GLP-1-based therapeutics in the pipeline.

Bombesin/oligoarginine fusion peptides for gastrin releasing peptide receptor (GRPR) targeted gene delivery.

The development of non-viral gene delivery systems, with the capacity to overcome most of the biological barriers facing gene delivery, is challenging. We have developed peptide-based, multicomponent, non-viral delivery systems, incorporating: a bombesin peptide ligand (BBN(6-14)), to selectively target the gastrin releasing peptide receptor (GRPR); oligoarginine peptides (hexa- (R6) and nona-arginine (R9)), for plasmid DNA (pDNA) condensation; and GALA, to facilitate endosome escape. The uptake and endosome escape efficiency of bombesin/oligoarginine and bombesin/oligoarginine/GALA fusion peptides for oligonucleotide delivery was evaluated in terms of their complex size, cellular uptake, endosome escape, and cellular toxicity. Complex size and cell uptake studies demonstrated that the nona-arginine/bombesin delivery system was more efficient at condensing and delivering pDNA into PC-3 prostate cancer cells compared to the hexa-arginine/bombesin delivery system. Further, competition with free bombesin peptide, and comparative uptake studies in Caco-2 cells, which express GRPR at a lower level, suggested that GRPR contributes to the targeted uptake of this system. The addition of GALA into the nona-arginine/bombesin-based system further increased the pDNA cellular uptake at all tested N/P ratios; facilitated endosomal pDNA release; and had limited effects on cell viability. In conclusion, the delivery system combining BBN(6-14) with nona-arginine and GALA had optimal characteristics for the delivery of pDNA into the GRPR overexpressing cell line PC-3.

Preparation of albendazole-loaded liposomes by supercritical carbon dioxide processing.

Supercritical fluid (SCF) technology offers a potential green alternative to organic solvent-based methods for drug formulation. Albendazole (ABZ) has promising anticancer activity when formulated to increase its cellular uptake. Herein, a static volume method was used to determine the solubility of ABZ in supercritical carbon dioxide (scCO2) for the future development of such ABZ formulations. The solubility of ABZ in scCO2 (250 bar, 37 °C) was approximately 12 mg/100 mL. The extent of dissolution was measured at various time points to determine when saturation solubility occurred, which was demonstrated from 9 h. In order to determine if scCO2 processing induced ABZ polymorphism, DSC/TGA, FTIR and XRD were used, which demonstrated no change in its solid state. Following this, ABZ loaded liposomes were manufactured using SCF technology. The liposomes diameter was 167.2 ± 5.3 nm as determined by Zetasizer, and confirmed by cryo-transmission electron microscopy. In conclusion, scCO2 was used successfully to solubilize ABZ, and to manufacture liposomes of nano-sized range. This study provides insight into use of green technology for future ABZ liposomal formulation without the need for organic solvents.

Multifunctional peptide-lipid nanocomplexes for efficient targeted delivery of DNA and siRNA into breast cancer cells.

The development of carriers for the delivery of oligonucleotide therapeutics is essential for the successful translation of gene therapies to the clinic. In the present study, a delivery system, which combines the fusogenic lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with a well-defined synthetic multifunctional peptide, was produced and optimized for gene delivery, with the aim to develop an efficient gene delivery platform for breast cancer cells. For this purpose, a breast cancer-specific cell targeting peptide (CTP) was incorporated into our leading peptide-based gene delivery system (to generate DEN-K(GALA)-TAT-K(STR)-CTP) to improve its cell-specific internalization, and investigated in combination with a formulation approach (DOPE/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)). DEN-K(GALA)-TAT-K(STR)-CTP alone efficiently complexed with DNA or siRNA, and promoted efficient cellular uptake, but low levels of gene expression. By adding the formulation approach, synergistic improvements in gene expression and silencing were observed compared to the peptide or formulation approaches alone. Of significance, this current system demonstrated more efficient gene knockdown when compared to the leading commercial siRNA delivery agent Lipofectamine® RNAiMAX. The utility of this system was demonstrated through the delivery of BCL2 (B-cell lymphoma 2) siRNA to MCF-7 cells, which led to near complete knockdown of the Bcl-2 protein, and inhibition of MCF-7 cell migration in a wound healing assay. The present peptide/lipid hybrid system is an excellent candidate for the delivery of DNA or siRNA into breast cancer cells. STATEMENT OF SIGNIFICANCE: The identification of safe and effective delivery systems for DNA and siRNA is of great importance. Herein, we developed a well-defined, multifunctional and cell-specific lipidic peptide DEN-K(GALA)-TAT-K(STR)-CTP as a breast cancer cell targeted gene delivery vector. When combined with a lipid component (DOTAP/DOPE), the peptide/lipid hybrid system demonstrated higher gene expression or knockdown levels compared to the peptide or lipid approach alone when used to deliver pDNA or siRNA respectively, indicating synergistic enhancement of gene delivery efficiency. Importantly, this delivery strategy achieved greater knockdown of the Bcl-2 protein when compared to the leading commercial siRNA delivery system Lipofectamine® RNAiMAX, suggesting its potential utility for the targeted treatment of Bcl-2 overexpressing breast cancers.

Design and evaluation of a stearylated multicomponent peptide-siRNA nanocomplex for efficient cellular siRNA delivery.

AIM: To develop a new synthetic peptide-based nanoparticulate siRNA delivery system. MATERIALS & METHODS: DEN-K(GALA)-TAT-K(STR) was generated by incorporating stearic acid into a multicomponent peptide (DEN-K(GALA)-TAT), containing a cationic poly-L-lysine dendron, an endosome-disrupting peptide GALA and a cell-penetrating peptide TAT(48-60). Its physicochemical characteristics, size, toxicity, cellular uptake and gene knockdown activity of the peptide/siRNA complexes were studied. RESULTS: DEN-K(GALA)-TAT-K(STR) exhibited a pH-responsive behavior, which assists with endosomal escape. When siRNA was delivered by DEN-K(GALA)-TAT-K(STR), it showed a significantly enhanced cellular uptake, compared with the nonlipidic peptide. This system also displayed enhanced knockdown efficiency and reduced cytotoxicity over the widely used delivery system branched 25-kDa polyethyleneimine. CONCLUSION: Our stearylated multicomponent delivery system has great potential as an efficient siRNA delivery vector.