Development of a dry powder for inhalation of nanoparticles codelivering cisplatin and ABCC3 siRNA in lung cancer.

Background: The current study sought to formulate a dry powder inhalant (DPI) for pulmonary delivery of lipopolymeric nanoparticles (LPNs) consisting of cisplatin and siRNA for multidrug-resistant lung cancer. siRNA against ABCC3 gene was used to silence drug efflux promoter. Results & discussion: The formulation was optimized through the quality by design system by nanoparticle size and cisplatin entrapment. The lipid concentration, polymer concentration and lipid molar ratio were selected as variables. The DPI was characterized by in vitro deposition study using the Anderson cascade impactor. DPI formulation showed improved pulmonary pharmacokinetic parameters of cisplatin with higher residence time in lungs. Conclusion: Local delivery of siRNA and cisplatin to the lung tissue resulted into an enhanced therapeutic effectiveness in combating drug resistance.

Co-delivery of cisplatin and siRNA through hybrid nanocarrier platform for masking resistance to chemotherapy in lung cancer.

The resistance of cancer cells to chemotherapy has presented a formidable challenge. The current research aims at evaluating whether silencing of the cisplatin efflux promoter gene ABCC3 using siRNA co-loaded with the drug in a nanocarrier improves its efficacy in non-small cell lung cancer (NSCLC). Hybrid nanocarriers (HNCs) comprising lipids and poly(lactic acid-polyethylene glycol) di-block copolymer (PEG-PLA) were prepared for achieving the simultaneous delivery of cisplatin caprylate and ABCC3-siRNA to the cancer cells. PEGylation of the formulated HNCs was carried out using post-insertion technique for imparting long circulation characteristics to the carrier. The optimized formulation exhibited an entrapment efficiency of 71.9 ± 2.2% and 95.83 ± 0.39% for cisplatin caprylate and siRNA respectively. Further, the HNC was found to have hydrodynamic diameter of 153.2 ± 1.76 nm and + 25.39 ± 0.49 mV zeta potential. Morphological evaluation using cryo transmission electron microscopy confirmed the presence of lipid bilayer surrounding the polymeric core in HNCs. The in vitro cellular uptake studies showed improved uptake, while cell viability studies of the co-loaded formulation in A549 cell-line indicated significantly improved cytotoxic potential when compared with drug solution and drug-loaded HNCs; cell cycle analysis indicated increased percentage of cell arrest in G2-M phase compared with drug-loaded HNCs. Further, the gene knock-down study showed that silencing of ABCC3 mRNA might be improved in vitro efficacy of the formulation. The optimized cisplatin and ABCC3 siRNA co-loaded formulation presented significantly increased half-life and tumour regression in A549 xenograft model in BALB/c nude mice. In conclusion, siRNA co-loaded formulation presented reduced drug resistance and increased efficacy, which might be promising for the current cisplatin-based treatments in NSCLC.

Synergistic co-loading of vincristine improved chemotherapeutic potential of pegylated liposomal doxorubicin against triple negative breast cancer and non-small cell lung cancer.

The current work aims to explore the biological characteristics of vincristine synergistic co-loading into pegylated liposomal doxorubicin in non-indicated modalities of non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC). The combinatorial liposome prepared by active co-loading of the drugs against modified ammonium ion gradient exhibited 95% encapsulation of both drugs. The cellular uptake studies using confocal microscopy and flow cytometry showed significantly increased uptake of dual drug formulation as against liposomal doxorubicin. The co-loaded liposome formulation had significantly increased cell cycle arrest in G2/M phase with subsequent apoptosis and reduced cell viability in both tumor cell lines than doxorubicin liposome. This carrier exhibited similar acute toxicity, pharmacokinetic and tissue distribution profiles with significant increase in tumor regression as compared to liposomal doxorubicin. These results indicate that co-encapsulation of vincristine into clinically used pegylated liposomal doxorubicin significantly improved in-vitro and in-vivo therapeutic efficacy against NSCLC and TNBC.

Optimization and efficacy study of synergistic vincristine coloaded liposomal doxorubicin against breast and lung cancer.

Aim: To improve the efficacy of poly-ethylene glycol (PEG)ylated liposomes coloaded with doxorubicin and vincristine against triple-negative breast cancer (TNBC) and non-small-cell lung cancer (NSCLC). Methods: The combinatorial index of the drugs was established using the Chou-Talalay method in MDA-MB-231 and A549 cell lines. The most effective ratio was co-encapsulated in factorial design optimized nanoliposomes which were characterized for similarity to clinical standard and evaluated in vitro and in vivo for therapeutic efficacy. Results & conclusion: The formulation exhibited more than 95% co-encapsulation, a size of 95.74 ± 2.65 nm and zeta potential of -9.17 ± 1.19 mV while having no significant differences in physicochemical and biochemical characteristics as compared with the clinical standard. Efficacy evaluation studies showed significantly improved cytotoxicity and tumor regression compared with liposomal doxorubicin indicating improvement in efficacy against TNBC and NSCLC.

Surface engineered liposomal delivery of therapeutics across the blood brain barrier: recent advances, challenges and opportunities.

Introduction: The delivery of drug payload to treat various brain diseases are met with various hindrances owing to the presence of the homeostasis regulatory gate, Blood-Brain Barrier (BBB). Although, pathogenesis and progression of the brain diseases alter the permeability of this barrier, effective delivery of agents is not achieved for the attainment of desired treatment outcomes. Liposomes with their salient properties have proven to be exciting options to navigate therapeutics across this barrier.Areas covered: This review tends to establish a correlation between the pathophysiology of disease affected barrier, with liposome-based passive and active delivery approaches for therapeutic agents, permitting their transport across the BBB. The potential of these carriers to present therapeutically effective agents' concentrations to the desired site of action have also been explored. Further, assessment of physicochemical, biopharmaceutical, and biological properties required for efficient translation of such carriers from bench to bedside has been made.Expert opinion: The encapsulation of the therapeutics in these structures enables suitable pro-brain delivery modifications of inherent pharmacokinetic-pharmacodynamic profiles along with appropriate surface engineering opportunities to deliver the drug cargo to the intended locations in the brain. However, a careful balance between the use of these surface-modified structures and toxicity potential needs to be ascertained for clinical safety and effectiveness.

An in vitro Assessment of Thermo-Reversible Gel Formulation Containing Sunitinib Nanoparticles for Neovascular Age-Related Macular Degeneration.

Anti-vascular endothelial growth factor agents have been widely used to treat several eye diseases including age-related macular degeneration (AMD). An approach to maximize the local concentration of drug at the target site and minimize systemic exposure is to be sought. Sunitinib malate, a multiple receptor tyrosine kinase inhibitor was encapsulated in poly(lactic-co-glycolic acid) nanoparticles to impart sustained release. The residence time in vitreal fluid was further increased by incorporating nanoparticles in thermo-reversible gel. Nanoparticles were characterized using TEM, DSC, FTIR, and in vitro drug release profile. The cytotoxicity of the formulation was assessed on ARPE-19 cells using the MTT assay. The cellular uptake, wound scratch assay, and VEGF expression levels were determined in in vitro settings. The optimized formulation had a particle size of 164.5 nm and zeta potential of - 18.27 mV. The entrapment efficiency of 72.0% ± 3.5% and percent drug loading of 9.1 ± 0.7% were achieved. The viability of ARPE-19 cells was greater than 90% for gel loaded, as such and blank nanoparticles at 10 µM and 20 µM concentration tested, whereas for drug solution viability was found to be 83% and 71% respectively at above concentration. The cell viability results suggest the compatibility of the developed formulation. Evaluation of cellular uptake, wound scratch assay, and VEGF expression levels for the developed formulations indicated that the formulation had higher uptake, superior anti-angiogenic potential, and prolonged inhibition of VEGF activity compared with drug solution. The results showed successful development of sunitinib-loaded nanoparticle-based thermo-reversible gel which may be used for the treatment of neovascular AMD.

Lipid-nucleic acid nanoparticles of novel ionizable lipids for systemic BMP-9 gene delivery to bone-marrow mesenchymal stem cells for osteoinduction.

Rational design of novel ionizable lipids for development of lipid-nucleic acid nanoparticles (LNP) is required for safe and effective systemic gene delivery for osteoporosis. LNPs require suitable characteristics for intravenous administration and effective accumulation in bone marrow for enhanced transfection. Hence, lipids with C18 tail and ionizable headgroup (Boc-His-ODA/BHODA and His-ODA/HODA) were synthesized and characterized physicochemically. LNPs were prepared with bone morphogenetic protein-9 gene (BHODA-LNP, HODA-LNP, and bone-homing peptide targeted HODA-LNP - HODA-LNPT). Thorough physicochemical (electrolyte stability, DNase I and serum stability) and biological (hemolysis, ROS induction, cytotoxicity and transfection) characterization was carried out followed by acute toxicity studies and therapeutic performance studies in ovariectomized rat model. Lipids with pH dependent ionization were successfully synthesized. LNPs thereof were ∼100 nm size with stability against electrolytes, DNase I and serum and exhibited low hemolytic potential demonstrating suitability for intravenous administration. LNPs exhibited minimal cytotoxicity, non-significant ROS induction and high transfection. In vivo studies demonstrated safety and improved bone regeneration in OVX rats with HODA-LNPT showing significantly better performance. Synthesized ionizable lipids offer safe and effective alternative for preparation of LNPs for gene delivery. Targeted BMP-9 LNP show potential for systemic osteoporosis treatment.

Combinatorial nanocarriers against drug resistance in hematological cancers: Opportunities and emerging strategies.

Hematological cancers are a group of malignancies affecting human hematopoietic and lymphoid tissues. Although the patients respond to treatment regimen during initial phases, the hematoma tumor heterogeneity results in the presence of some minimal disease residue thereby exhibiting remission, relapses or refractoriness in disease conditions leading to poor overall survival period. The current therapeutic standard practices involve blending of conventional agents with novel targeting agents or immune-therapeutics in a cocktail to effectively reap the benefits of drugs acting through multiple signaling pathways. Considerable evaluation of the risk benefit ratio on part of clinicians is necessitated to select the best optimum therapy considering the high incidences of drug resistance. This drug resistance may be attributed to faulty upregulation or mutation of multiple drug resistance regulating genes, increased tumor cell immune system cross talk, increased expression of drug efflux pump inducers and inhibition of apoptosis among others. Conventional single drug nanotherapeutics as modulators of drug resistance have already clinically exhibited their potential by passively delivering the active cargo to desired targets in hematological neoplasms. However, with the ever-growing clinical failures of such therapies, the landscape of hematological cancer treatment has seen a plethora of changes in the last few years. The two towering changes in the treatment has been the approval of combinatorial drug nanocarrier Vyxeos™ and chimeric antigen receptor T cell (CAR-T) therapy Kymriah™ as well as Yescarta™. The approval of CAR-T therapy not only resulted in a paradigm shift in the avenues of blood cancer treatment towards personalized approaches but also saddled it with questions of economic viability and effectiveness in the entire spectrum of such neoplasms. Under such conditions, combinatorial drug nanocarriers encompassing synergistic ratios of clinically effective drug combinations affording temporal and spatial control present an exciting approach to overcome these drug resistance modalities. This platform provides increased chances of therapeutic in-vitro in-vivo correlation along with minimization of drug resistance and associated disease relapse conditions. The present review intends to present the current preclinical and clinical advances in combinatorial nanocarrier mediated management of drug resistance in hematological cancers.

Colloidally Stable Small Unilamellar Stearyl Amine Lipoplexes for Effective BMP-9 Gene Delivery to Stem Cells for Osteogenic Differentiation.

The biocompatibility of cationic liposomes has led to their clinical translation in gene delivery and their application apart from cancer to cardiovascular diseases, osteoporosis, metabolic diseases, and more. We have prepared PEGylated stearyl amine (pegSA) lipoplexes meticulously considering the physicochemical properties and formulation parameters to prepare single unilamellar vesicles (SUV) of < 100 nm size which retain their SUV nature upon complexation with pDNA rather than the conventional lipoplexes which show multilamellar nature. The developed PEGylated SA lipoplexes (pegSA lipoplexes) showed a lower N/P ratio (1.5) for BMP-9 gene complexation while maintaining the SUV character with a unique shape (square and triangular lipoplexes). Colloidal and pDNA complexation stability in the presence of electrolytes and serum indicates the suitability for intravenous administration for delivery of lipoplexes to bone marrow mesenchymal stem cells through sinusoidal vessels in bone marrow. Moreover, lower charge density of lipoplexes and low oxidative stress led to lower toxicity of lipoplexes to the C2C12 cells, NIH 3T3 cells, and erythrocytes. Transfection studies showed efficient gene delivery to C2C12 cells inducing osteogenic differentiation through BMP-9 expression as shown by enhanced calcium deposition in vitro, proving the potential of lipoplexes for bone regeneration. In vivo acute toxicity studies further demonstrated safety of the developed lipoplexes. Developed pegSA lipoplexes show potential for further in vivo preclinical evaluation to establish the proof of concept.

Lipid-Based Oral Formulation Strategies for Lipophilic Drugs.

Partition coefficient (log P) is a key physicochemical characteristic of lipophilic drugs which plays a significant role in formulation development for oral administration. Lipid-based formulation strategies can increase lymphatic transport of these drugs and can enhance bioavailability many folds. The number of lipophilic drugs in pharmacopoeias and under discovery are continuously increasing and making the job of the formulation scientist difficult to develop suitable formulation of these drugs due to potent nature and water insolubility of these drugs. Recently, many natural and synthetic lipids are appearing in the market which are helpful in the development of lipid-based formulations of these types of drugs having enhanced solubility and bioavailability. One such reason for this enhanced bioavailability is the accessibility of the lymphatic transport as well as avoidance of first-pass effect. This review discusses the impact of lipophilicity in enhancing the intestinal lymphatic drug transport thereby reducing first-pass metabolism. The most appropriate strategy for developing a lipid-based formulation depending upon the degree of lipophilicity has been critically discussed and provides information on how to develop optimum formulation. Various formulation strategies are discussed in-depth by classifying lipid-based oral drug delivery systems with case studies of few marketed formulations with challenges and opportunities for the future of the formulations.

Antimicrobial peptide delivery: an emerging therapeutic for the treatment of burn and wounds.

The management of wounds and burns is becoming difficult using conventional therapeutics available due to resistance development by microbes. Therefore, there is an utmost need to develop therapeutic alternatives to these agents. Antimicrobial peptides have emerged as a novel class of agents for the effective management of wounds and burns due to their potent nature along with minimal chances of resistance development against them. This article focuses on highlighting the importance of these antimicrobial peptides among the various therapeutic alternatives for burns and wounds. Further, effective delivery strategies for these agents that are being employed and investigated are reported along with an overview of the importance of these agents in the coming years.

Liposomes encapsulating native and cyclodextrin enclosed paclitaxel: Enhanced loading efficiency and its pharmacokinetic evaluation.

Combination strategy involving cyclodextrin (CD) complexation and liposomal system was investigated for Paclitaxel (PTX) to improve loading. Complexation was done using 2,6-di-O-methylbetacyclodextrin (DMßCD). Sterically stabilized double loaded PEGylated liposomes (DLPLs) containing PTX and PTX-DMßCD complex were prepared by thin film hydration. Physicochemical characterization of complex and prepared DLPLs was carried out. Cytotoxic potential, hemolytic potential and pharmacokinetics of DLPLs were tested in comparison to Taxol®. Aqueous solubility of PTX increased by almost 3 × 104 folds due to complexation with DMßCD as compared to pure drug solubility. Liposomal system was found to have 162.8 ± 4.1 nm size, zeta potential of -5.6 ± 0.14 mV and 2-fold increase in drug loading to 5.8 mol % for PTX due to double loading. DLPLs had low hemolytic potential and higher cytotoxicity on SKOV3 cells with improvement in IC50 value by 4.2 folds as compared to Taxol® at 48 h. The anti-angiogenic potential of DLPLs was confirmed by 1.33 folds lesser wound recovery in SKOV3 cells compared to Taxol®. In-vivo pharmacokinetic evaluation of DLPLs in rats substantiates improvement in circulation time, higher plasma concentration and decreased clearance rate compared to Taxol®. An efficacious system with improved loading and pharmacokinetics was formulated as potential alternative for currently marketed PTX formulation.

Hydroxyethyl substituted linear polyethylenimine for safe and efficient delivery of siRNA therapeutics.

Linear polyethylenimine (LPEI) has been well reported as a carrier for siRNA delivery. However, its applications are limited due to its highly ionized state at physiologic pH and the resultant charge mediated toxicity. The presence of ionizable secondary amines in LPE are responsible for its unique characteristics such as pH dependent solubility and positive charge. Therefore, modification of LPEI was carried out to obtain hydroxyethyl substituted LPEI with the degree of substitution ranging from 15% to 45%. The impact of modification on the physicochemical parameters of the polymer, i.e. buffer capacity, solubility, biocompatibility and stability, was evaluated. Surprisingly, despite the loss of ionizable amines, the substitution improved solubility, and even overcame the pH dependent solubility of LPEI. In addition, the conversion of secondary amines to less basic tertiary amines after substitution improved the buffer capacity, in the endosomal pH range, required for efficient endosomal escape. It also reduced erythrocyte aggregation, hemolytic potential and in vitro cytotoxicity. The in vitro studies showed enhanced cell uptake and mRNA knockdown efficiency. Thus, the proposed modification shows a simple approach to overcome the limitation of LPEI for siRNA delivery.

Challenges in Dermal Delivery of Therapeutic Antimicrobial Protein and Peptides.

BACKGROUND: Protein and peptides in biological system form an important part of innate immune system and are being explored for potential use in various diseases as therapeutics. Importance of proteins and peptides as a new class of antimicrobial agents has boosted research in the field of biotechnology as potential alternative to antibiotic agents. OBJECTIVE: Protein and peptides antimicrobial as a therapeutic class are structurally diverse and exhibit potent activity against microbes by various mechanisms. However, they present formidable challenge in formulation due to requirement of specific spatial configuration for their activity and stability. Thus, encapsulation of these therapeutics in various nano-systems may sustain activity along with improvement in stability. METHOD: The article highlights the need for antimicrobial peptides in dermal infections along with discussion of mechanism of their action. It highlights challenges faced for dermal delivery and research carried out for their successful delivery using nano-systems. RESULTS: It is widely realized that these novel classes of therapeutic agents have tremendous market potential to emerge as an alternative to conventional antibiotic agents for combating issue of multidrug resistant microbial species. Research in their delivery aspects by use of current advances made in delivery systems through use of nanoconstructs offers much needed area for exploration and achieving success. CONCLUSION: As there is an urgent need for coming up with new therapeutic agents for encompassing the increased burden of microbial diseases in human population as well as their delivery challenges, research in field will give the much-needed strategic advantage against pathogenic organisms.

Approaches and Recent Trends in Gene Delivery for Treatment of Atherosclerosis.

BACKGROUND: The development and progression of atherosclerosis is known to occur at a sluggish pace and the lesions remain concealed for a long duration before the factual situation of the complex atherosclerotic etiology affecting various organ gets apprehended. The root cause mainly involves an imbalance or malfunction of the cholesterol metabolizing pathway. The till date therapeutic alternatives include oral hypo-lipidemic agents along with advances made in biotechnology/tissue engineering and surgical procedures for management purpose. However, with the advent and upsurge of nanotech delivery systems, along with meticulous indulgence and identification of the causative genes in the etiology of disease have opened a new therapeutic area that has far reaching application potential for effective management of such chronic disease requiring lifelong therapy. METHODS: Various genes that have implication in atherosclerosis were reviewed along with research in delivery vectors that have been employed for gene therapeutics and hurdles in successful delivery were elaborated. Relevant patents are discussed systematically to clearly support and highlight the developments made. RESULTS: Patenting activity in the delivery of genes for atherosclerosis so far primarily covers use of viral vectors. With the identification of new targets, a list of candidate genes are available that can be potentially exploited for therapeutic purpose. Though the delivery of candidate genes using viral vectors has been well explored, the limitation of viral vectors have seized the much needed clinical success. Non-viral vectors can prove to be the key for conquering this limitations and offer a vast area for exploration for achieving an effective control and remission to the disease and increasing the assortment of patents as reviewed in this article. CONCLUSION: In view of the many limitations in employing viral vectors for delivery, designing non-viral vectors for successful delivery of therapeutic gene in atherosclerosis should be realized and focused for effective management of the disease.

Role of Nanotechnology in Delivery of Protein and Peptide Drugs.

The advent of recombinant DNA technology and computational designing has fueled the emergence of proteins and peptides as a new class of modern therapeutics such as vaccines, antigens, antibodies and hormones. Demand for such therapeutics has increased recently due to their distinct pharmacodynamic characteristics of specificity of action and high potency. However, their potential clinical applications are often hindered by involvement of factors which impact their therapeutic potential negatively. Large size, low permeability, conformational fragility, immunogenicity, metabolic degradation and short half-life results in poor bioavailability and inferior efficacy. These challenges have encouraged researchers to devise strategies for effective delivery of proteins and peptides. Recent advances made in nanotechnology are being sought to overcome aforesaid problems and to offer advantages such as higher drug loading, improved stability, sustained release, amenability for non-parenteral administration and targeting through surface modifications. This review focuses on elaborating the role of nanotechnology based formulations and associated challenges in protein and peptide delivery, their clinical outlook and future perspective.