Development and validation of an ultrahigh-performance liquid chromatography-tandem mass spectrometry method to investigate the plasma pharmacokinetics of a KCa 2.2/KCa 2.3 positive allosteric modulator in mice.

RATIONALE: There is currently no treatment for spinocerebellar ataxias (SCAs), which are a group of genetic disorders that often cause a lack of coordination, difficulty walking, slurred speech, tremors, and eventually death. Activation of KCa 2.2/KCa 2.3 channels reportedly exerts beneficial effects in SCAs. Here, we report the development and validation of an analytical method for quantitating a recently developed positive allosteric modulator of KCa 2.2/KCa 2.3 channels (compound 2q) in mouse plasma. METHODS: Mouse plasma samples (10 µL) containing various concentrations of 2q were subjected to protein precipitation in the presence of a structurally similar internal standard (IS). Subsequently, the analytes were separated on a C18 ultrahigh-performance liquid chromatography column and detected by a tandem mass spectrometer. The method was validated using US Food and Drug Administration (FDA) guidelines. Finally, the validated assay was applied to the measurement of the plasma concentrations of 2q in plasma samples taken from mice after single intravenous doses of 2 mg/kg of 2q, and the pharmacokinetic parameters of 2q were determined. RESULTS: The calibration standards were linear (r2 ≥ 0.99) in the range of 1.56-200 nM of 2q with intra- and inter-run accuracy and precision values within the FDA guidelines. The lower limit of quantitation of the assay was 1.56 nM (0.258 pg on the column). The recoveries of 2q and IS from plasma were >94%, with no appreciable matrix effect. The assay showed no significant carryover, and the plasma samples stored at -80°C or the processed samples stored in the autosampler at 10°C were stable for at least 3 weeks and 36 h, respectively. After intravenous injection, 2q showed a bi-exponential decline pattern in the mouse plasma, with a clearance of 30 mL/min/kg, a terminal volume of distribution of 1.93 mL/kg, and a terminal half-life of 45 min. CONCLUSIONS: The developed assay is suitable for preclinical pharmacokinetic-pharmacodynamic studies of 2q as a potential drug candidate for ataxias.

Redox-Responsive Disulfide Cyclic Peptides: A New Strategy for siRNA Delivery.

RNA interference (RNAi) is a powerful tool capable of targeting virtually any protein without time-consuming and expensive drug development studies. However, due to obstacles facing efficient and safe delivery, RNAi-based therapeutic approach remains a challenge. Herein, we have designed and synthesized a number of disulfide-constraining cyclic and hybrid peptides using tryptophan and arginine residues. Our hypothesis was that peptide structures would undergo reduction by intracellular glutathione (more abundant in cancer cells) and unpack the small interfering RNA (siRNA) from the peptide/siRNA complexes. A subset of newly developed peptides (specifically, C4 and H4) exhibited effective cellular internalization of siRNA (∼70% of the cell population; monitored by flow cytometry and confocal microscopy), the capability of protecting siRNA against early degradation by nucleases (monitored by gel electrophoresis), minimal cytotoxicity in selected cell lines (studied by cell viability and LC50 calculations), and efficient protein silencing by 70-75% reduction in the expression of targeting signal transducer and activator of transcription 3 (STAT3) in human triple-negative breast cancer (TNBC) MDA-MB-231 cells, analyzed using the Western blot technique. Our results indicate the birth of a promising new family of siRNA delivery systems that are capable of safe and efficient delivery, even in the presence of nucleases.

Single and Combinational siRNA Therapy of Cancer Cells: Probing Changes in Targeted and Nontargeted Mediators after siRNA Treatment.

Cancer cells are known to be heterogeneous and plastic, which imparts innate and acquired abilities to resist molecular targeting by short interfering RNA (siRNA). Not all cancer cells in a population would show a similar responsiveness to targeting of genes critical for their survival and even the responders could quickly transform and switch to alternative mechanism(s) for their survival. This study was designed to look at this phenomenon by analyzing the effect of siRNA silencing of selected protein mRNAs involved in cell survival and proliferation on other protein mRNAs that could contribute to cell survival. We compared the gene expression profile of the initial population after siRNA silencing to the subpopulation that survived the siRNA silencing, to identify potential overexpressions that might explain the cell survival. Our studies show that silencing well-selected protein mRNAs simultaneously could offer advantages compared to individual siRNA silencing due to an additional impact on the expression level of certain protein mRNAs. We also demonstrate that overexpression of certain protein mRNAs could explain the innate unresponsiveness of a subpopulation of cells. These observations could be a stepping stone for further investigation of the possibility of significant synergistic effect for this combinational RNA interference strategy.

Effect of siRNA pre-Exposure on Subsequent Response to siRNA Therapy.

PURPOSE: An alternative cancer therapy based on RNA interference (RNAi) has shown considerable promise but the possibility of resistance development is not known. This study explored the possibility of therapeutic resistance against siRNA nanoparticles in human cancer cells. METHODS: Two approaches to siRNA treatment were undertaken using lipid-modified polyethylenimines, a single high concentration (shock) and repeated increasing concentrations (gradual). The targets were Mcl-1, RPS6KA5 and KSP in MDA-MB-435 cells. RESULTS: There was no evidence of resistance development in shock-treated cells, while the decrease in mRNA levels of targeted proteins was not as robust in naïve cells in gradual treatment. However, silencing efficiency was restored after a 7-day recovery period when expression of suppressed proteins returned to normal levels. Cellular uptake of siRNA was not affected by pre-treatments. Other mediators involved in cell survival and proliferation were altered in siRNA-treated cells, but only JUN silencing led to a heightened loss of viability. In vivo experiments demonstrated similar silencing efficiency at mRNA level after repeat doses. CONCLUSIONS: Human cancer cells responded to repeat siRNA nanoparticles in a similar fashion after a temporary initial alteration and little, if any, resistance was evident against repeated siRNA treatments.

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

[(WR)8WKßA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin.

Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKßA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKßA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 µM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 µM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKßA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 µM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKßA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it.

siRNA-mediated down-regulation of P-glycoprotein in a Xenograft tumor model in NOD-SCID mice.

PURPOSE: The efficacy of chemotherapy is decreased due to over-expression of the drug transporter P-glycoprotein (P-gp). This study was conducted to determine the feasibility of down-regulating tumor P-gp levels with non-viral siRNA delivery in order to sensitize the tumors to drug therapy. METHODS: P-gp over-expressing MDA435/LCC6 MDR1 cells were used to establish xenografts in NOD-SCID mouse. Cationic polymers polyethylenimine (PEI) and stearic acid-substituted poly-L-lysine (PLL-StA) were formulated with P-gp- specific siRNAs and delivered intratumorally to explore the feasibility of P-gp down-regulation in tumors. Intravenous Doxil™ was administered to investigate tumor growth. RESULTS: PEI and PLL-StA effectively delivered siRNA to MDA435/LCC6 MDR1 cells in vitro to reduce P-gp expression for 3 days. Intratumoral injection of siRNA with the carriers resulted in 60-80% and 20-32% of siRNA retention in tumors after 24 and 96 hr, respectively. This led to ~29.0% and ~61.5% P-gp down-regulation with PEI- and PLL-StA-mediated siRNA delivery, respectively. The P-gp down-regulation by intratumoral siRNA injection led to better response to systemic Doxil™ treatment, resulting in slowed tumor growth in originally doxorubicin-resistant tumors. CONCLUSION: Effective P-gp down-regulation was feasible with polymeric siRNA delivery in a xenograft model, resulting in an enhanced response to the drug therapy.

Design and application of hybrid cyclic-linear peptide-doxorubicin conjugates as a strategy to overcome doxorubicin resistance and toxicity.

Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C-S-S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 µM after 72 h of incubation, which was comparable to Dox (5 µM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2, <9%) at concentrations of 5-10 µM (compared to free Dox at 5 µM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 µM) in H9C2 cells. In comparison, Dox (5 µM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 µM, respectively) compared to free Dox (LC50 of 36-41 µM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.

Prospects for RNAi Therapy of COVID-19.

COVID-19 caused by the SARS-CoV-2 virus is a fast emerging disease with deadly consequences. The pulmonary system and lungs in particular are most prone to damage caused by the SARS-CoV-2 infection, which leaves a destructive footprint in the lung tissue, making it incapable of conducting its respiratory functions and resulting in severe acute respiratory disease and loss of life. There were no drug treatments or vaccines approved for SARS-CoV-2 at the onset of pandemic, necessitating an urgent need to develop effective therapeutics. To this end, the innate RNA interference (RNAi) mechanism can be employed to develop front line therapies against the virus. This approach allows specific binding and silencing of therapeutic targets by using short interfering RNA (siRNA) and short hairpin RNA (shRNA) molecules. In this review, we lay out the prospect of the RNAi technology for combatting the COVID-19. We first summarize current understanding of SARS-CoV-2 virology and the host response to viral entry and duplication, with the purpose of revealing effective RNAi targets. We then summarize the past experience with nucleic acid silencers for SARS-CoV, the predecessor for current SARS-CoV-2. Efforts targeting specific protein-coding regions within the viral genome and intragenomic targets are summarized. Emphasizing non-viral delivery approaches, molecular underpinnings of design of RNAi agents are summarized with comparative analysis of various systems used in the past. Promising viral targets as well as host factors are summarized, and the possibility of modulating the immune system are presented for more effective therapies. We place special emphasis on the limitations of past studies to propel the field faster by focusing on most relevant models to translate the promising agents to a clinical setting. Given the urgency to address lung failure in COVID-19, we summarize the feasibility of delivering promising therapies by the inhalational route, with the expectation that this route will provide the most effective intervention to halt viral spread. We conclude with the authors' perspectives on the future of RNAi therapeutics for combatting SARS-CoV-2. Since time is of the essence, a strong perspective for the path to most effective therapeutic approaches are clearly articulated by the authors.

A systematic comparison of lipopolymers for siRNA delivery to multiple breast cancer cell lines: In vitro studies.

Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells.

Disposition of drugs in block copolymer micelle delivery systems: from discovery to recovery.

Since their discovery in the early 1980s, polymeric micelles have been the subject of several studies as delivery systems that can potentially improve the therapeutic performance and modify the toxicity profile of encapsulated drugs by changing their pharmacokinetic characteristics. The efforts in this area have led in recent years to the advancement of several polymeric micellar formulations to clinical trials, some of which have shown promise in changing the biodistribution of the incorporated drug after intravenous administration as a means of tumour-targeted drug delivery. Recently, the possible benefit of polymeric micellar delivery in enhancing the absorption and bioavailability of incorporated drugs from alternative routes of drug administration has attracted interest. This article provides an overview of the effect of polymeric micellar delivery on absorption, distribution, metabolism and excretion of incorporated therapeutic agents. It also aims to assess the current information on the performance of polymeric micellar delivery systems in modifying the pharmacokinetics/pharmacodynamics of the incorporated drugs in clinical trials, and to re-examine the important structural factors required for successful design of polymeric micellar delivery systems capable of inducing favourable changes in the pharmacokinetics of the encapsulated drug.

Polymeric micellar delivery reduces kidney distribution and nephrotoxic effects of Cyclosporine A after multiple dosing.

The aim of this study was to test the ability of poly(ethylene oxide)-b-poly (epsilon-caprolactone) (PEO-b-PCL) micelles to reduce the renal uptake and nephrotoxicity of Cyclosporine A (CyA) after multiple dose administration. Sprague-Dawley rats received CyA i.v. at a dose of 20 mg/kg/day delivered as the commercial formulation (Sandimmune) or polymeric micellar formulation (PM-CyA). Cremophor EL (the solubilizing agent in Sandimmune), unloaded PEO-b-PCL micelles, or normal saline were also administered i.v. to control rats. After 7 days, kidney function was assessed through measurement of creatinine (CLcr) and urea clearances, as well as electrolyte concentrations in plasma. Blood and kidney were collected and assayed for CyA. Sandimmune administration led to decreased CLcr, and increased urea and potassium levels in plasma. In contrast, functional nephrotoxicity with the PM-CyA was not apparent, as the CLcr did not change significantly. The rate of increase in body weight in control rats was 3.1-3.4% per day. Weight gains (1.8% per day) were also noted in the rats given PM-CyA, although the body weight of animals receiving Sandimmune remained constant. Compared to Sandimmune, polymeric micelles reduced kidney uptake of CyA by 2.6-fold, and increased CyA levels in blood by 2.1-fold. The results show a potential for PEO-b-PCL micelles in restricting the nephrotoxicity of CyA.

Nanomedicine for immunosuppressive therapy: achievements in pre-clinical and clinical research.

INTRODUCTION: Immunosuppression is the mainstay therapy in organ transplantation and autoimmune diseases. The effective clinical application of immunosuppressive agents has suffered from the emergence of systemic immunosuppression and/or individual drug side effects. Nanotechnology approaches may be used to modify the mentioned shortcomings by enhancing the delivery of immunosuppressants to target cells of the immune system, thus reducing the required dose for function, and/or reducing drug distribution to non-target tissues. AREAS COVERED: We provide an overview on the development of nanotechnology products for the most commonly used immunosuppressive agents. At first, the rationale for the use of nanoparticles as means for immunosuppressive therapy is discussed. This is followed by a review of major accomplishments in this area, particularly in preclinical in vivo studies. EXPERT OPINION: The results of research conducted in this area to date, points to a great promise for nano-medicine in increasing the bioavailability, reducing the toxicity, and/or potentiating the activity of immunosuppressive agents. It is, therefore, safe to speculate the more rapid translation of nanotechnologyin clinical immunosuppressive therapy in the near future providing to the overcoming of hurdles associated with nano-drug delivery such as high cost, inadequate reproducibility and potential safety concerns of the delivery systems themselves.

Synthesis, characterization, and in vitro cytotoxicity of fatty acyl-CGKRK-chitosan oligosaccharides conjugates for siRNA delivery.

In this studies, three fatty acyl derivatives of CGKRK homing peptides were coupled successfully to chitosan oligosaccharides (COS) using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate sodium salt (sulfo-SMCC). The COS-SMCC was prepared by direct coupling between COS and sulfo-SMCC in PBS (pH7.5) at RT for 48h. The structure of COS-SMCC and the three fatty acyl-CGKRK-SMCC-COS conjugates were characterized by FT-IR, 13C NMR, and SEM. The ability of three conjugates to condense siRNA into nanosized polyplexes and their efficacy in protecting siRNA from serum nucleases degradation were investigated. Among the investigated derivatives, S-CGKRK-COS showed higher siRNA binding affinity as compared to the P-CGKRK-COS and O-CGKRK-COS, respectively. At a ratio of 10:1, complete protection for siRNA from early enzymatic degradation was achieved. The polymers and the polymer/siRNA polyplexes showed negligible cytotoxicity on human breast cancer cell line MDA-MB-231 at all investigated ratios. However, the polyplexes prepared with palmitoyl and oleoyl derivatives at polymer concentration 10µg/mL reduced the cell viability by 21.5% and 35%, respectively. The results of this study revealed the potential use of fatty acyl-CGKRK-COS as a siRNA carrier and confirmed the importance of incorporating a hydrophobic moiety into chitosan to improve its capacity in complexing with siRNA and protection from degradation.

Polymeric micelles for drug targeting.

Polymeric micelles are nano-delivery systems formed through self-assembly of amphiphilic block copolymers in an aqueous environment. The nanoscopic dimension, stealth properties induced by the hydrophilic polymeric brush on the micellar surface, capacity for stabilized encapsulation of hydrophobic drugs offered by the hydrophobic and rigid micellar core, and finally a possibility for the chemical manipulation of the core/shell structure have made polymeric micelles one of the most promising carriers for drug targeting. To date, three generations of polymeric micellar delivery systems, i.e. polymeric micelles for passive, active and multifunctional drug targeting, have arisen from research efforts, with each subsequent generation displaying greater specificity for the diseased tissue and/or targeting efficiency. The present manuscript aims to review the research efforts made for the development of each generation and provide an assessment on the overall success of polymeric micellar delivery system in drug targeting. The emphasis is placed on the design and development of ligand modified, stimuli responsive and multifunctional polymeric micelles for drug targeting.

Encapsulation of hydrophobic drugs in polymeric micelles through co-solvent evaporation: the effect of solvent composition on micellar properties and drug loading.

This study was designed to develop an optimized co-solvent evaporation procedure for the efficient encapsulation of hydrophobic drugs in polymeric micelles of methoxy poly(ethylene oxide)-block-poly(epsilon-caprolactone) (MePEO-b-PCL). MePEO-b-PCL block copolymers having varied MePEO and PCL molecular weights were synthesized, assembled to polymeric micelles, and used for the encapsulation of cyclosporine A (CyA) by a co-solvent evaporation method. The co-solvent composition was varied by changing the type of organic co-solvent (using acetone, acetonitrile and tetrahydrofuran), the ratio of organic to aqueous phase, and their order of addition. Carrier size, morphology and encapsulated CyA levels were defined by dynamic light scattering (DLS), transmission electron microscopy (TEM) and HPLC, respectively, and the effect of co-solvent composition on micellar properties and loaded CyA levels was evaluated. Application of acetone and acetonitrile as the selective co-solvent for the core-forming block led to a decrease in the average diameter of self-assembled structures. When acetone was added to water, a decrease in the ratio of organic to aqueous phase led to an increase in the loading efficiency of CyA in MePEO-b-PCL micelles. A similar trend in CyA loading was observed for MePEO-b-PCL micelles of varied MePEO and PCL block lengths. The ratio of organic to aqueous phase did not affect CyA loading when water was added to acetone. Irrespective of the order of addition, the decrease in the organic to aqueous phase ratio caused a reduction in the average diameter of the empty and CyA loaded micelles. We conclude that the co-solvent evaporation method may be optimized to improve the efficiency of drug encapsulation in polymeric micelles. For CyA encapsulation in MePEO-b-PCL micelles, addition of acetone to water at lower organic to aqueous phase ratio is shown to be the optimum procedure leading to higher drug encapsulation and smaller average diameter for the self-assembled structures.

Tumor-targeted delivery of siRNA using fatty acyl-CGKRK peptide conjugates.

Tumor-targeted carriers provide efficient delivery of chemotherapeutic agents to tumor tissue. CGKRK is one of the well-known tumor targeting peptides with significant specificity for angiogenic blood vessels and tumor cells. Here, we designed fatty acyl conjugated CGKRK peptides, based on the hypothesis that hydrophobically-modified CGKRK peptide could enhance cellular permeation and delivery of siRNA targeted to tumor cells for effective silencing of selected proteins. We synthesized six fatty acyl-peptide conjugates, using a diverse chain of saturated and unsaturated fatty acids to study the efficiency of this approach. At peptide:siRNA weight/weight ratio of 10:1 (N/P ≈ 13.6), almost all the peptides showed complete binding with siRNA, and at a w/w ratio of 20:1 (N/P ≈ 27.3), complete protection of siRNA from early enzymatic degradation was observed. Conjugated peptides and peptide/siRNA complexes did not show significant cytotoxicity in selected cell lines. The oleic acid-conjugated peptide showed the highest efficiency in siRNA uptake and silencing of kinesin spindle protein at peptide:siRNA w/w ratio of 80:1 (N/P ≈ 109). The siRNA internalization into non-tumorigenic kidney cells was negligible with all fatty acyl-peptide conjugates. These results indicate that conjugation of fatty acids to CGKRK could create an efficient delivery system for siRNA silencing specifically in tumor cells.

Polymeric micelles for drug delivery.

Polymeric micelles have been the subject of many studies in the field of drug delivery for the past two decades. The interest has specifically been focused on the potential application of polymeric micelles in three major areas in drug delivery: drug solubilisation, controlled drug release and drug targeting. In this context, polymeric micelles consisting of poly(ethylene oxide)-b-poly(propylene oxide), poly(ethylene oxide)-b-poly(ester)s and poly(ethylene oxide)-b-poly(amino acid)s have shown a great promise and are in the front line of development for various applications. The purpose of this manuscript is to provide an update on the current status of polymeric micelles for each application and highlight important parameters that may lead to the development of successful polymeric micellar systems for individual delivery requirements.

Identification of Potential Drug Targets in Cancer Signaling Pathways using Stochastic Logical Models.

The investigation of vulnerable components in a signaling pathway can contribute to development of drug therapy addressing aberrations in that pathway. Here, an original signaling pathway is derived from the published literature on breast cancer models. New stochastic logical models are then developed to analyze the vulnerability of the components in multiple signalling sub-pathways involved in this signaling cascade. The computational results are consistent with the experimental results, where the selected proteins were silenced using specific siRNAs and the viability of the cells were analyzed 72 hours after silencing. The genes elF4E and NFkB are found to have nearly no effect on the relative cell viability and the genes JAK2, Stat3, S6K, JUN, FOS, Myc, and Mcl1 are effective candidates to influence the relative cell growth. The vulnerabilities of some targets such as Myc and S6K are found to vary significantly depending on the weights of the sub-pathways; this will be indicative of the chosen target to require customization for therapy. When these targets are utilized, the response of breast cancers from different patients will be highly variable because of the known heterogeneities in signaling pathways among the patients. The targets whose vulnerabilities are invariably high might be more universally acceptable targets.