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.

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.

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.

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.

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.

PEGylated composite nanoparticles of PLGA and polyethylenimine for safe and efficient delivery of pDNA to lungs.

Achieving stable, efficient and non-toxic pulmonary gene delivery is most challenging requirement for successful gene therapy to lung. Composite nanoparticles (NPs) of the poly(lactic-co-glycolic acid) (PLGA) and cationic polymer polyethyleneimine (PEI) is an efficient alternative to viral and liposomal vectors for the pulmonary delivery of pDNA. NPs with different weight ratios (0-12.5%w/w) of PLGA/PEI were prepared and characterized for size, morphology, surface charge, pDNA loading and in vitro release. The in vitro cell uptake and transfection studies in the CFBE41o-cell line revealed that NPs with 10% w/w PEI were more efficient but they exhibited significant cytotoxicity in MTT assays, challenging the safety of this formulation. Surface modifications of these composite NPs through PEGylation reduced toxicity and enhanced cellular uptake and pDNA expression. PEGylation improved diffusion of NPs through the mucus barrier and prevented uptake by pulmonary macrophages. Finally, PEGylated composite NPs were converted to DPI by lyophilization and combined with lactose carrier particles, which resulted in improved aerosolization properties and lung deposition, without affecting pDNA bioactivity. This study demonstrates that a multidisciplinary approach may enable the local delivery of pDNA to lung tissue for effective treatment of deadly lung diseases.

Role of antibodies in diagnosis and treatment of ovarian cancer: Basic approach and clinical status.

Ovarian cancer is the second most leading gynecological cancer after endometrial cancer in women. Chemotherapy and cyto-reductive surgery are currently the mainstays for treatment of ovarian cancer in early stages. However, the overall 5years of survival rate in advanced stage is just 20-30%. The main reasons behind therapeutic failures and a low survival rate are challenges in early diagnosis, frequent recurrence, chemo-resistance development and lack of targeting. Antibodies have evolved as a rationale therapeutic approach to overcome these hurdles and to engender significant clinical applications. Detection of cancer associated antibodies and radiolabeled antibody conjugates are forming the basis for early diagnosis of ovarian cancer. Besides this, antibodies when given alone act as an anti-angiogenic agent or utilize the protective role of immune system against ovarian cancer. This high specificity and selectivity of action is also accompanied by development of tumor antigen-specific memory T cells, which can prevent cancer recurrence. In addition, when given in combination with chemotherapy, antibodies have turned out to sensitize chemo-resistant tumors. Antibodies are also playing cardinal role in design of highly potent class of targeted therapies, such as antibody-drug conjugates and clinically viable tumor targeted drug nanocarriers. This article aims to explore the fundamentals in development of antibody based therapeutics and multitude ways of clinical applications in diagnosis and treatment of ovarian cancer. Focus is given on the recent advances made in preclinical and clinical settings with an overview to unmet challenges so as to develop better immunotherapeutics in future.

Oral Absorption Promoters: Opportunities, Issues, and Challenges.

Transport of a drug across the biological membrane of the gastrointestinal tract has turned out to be a critical barrier against the success of any oral drug delivery technology. The unique advantages of the oral route, along with need for an oral substitute of invasive parenteral formulations and the reduction of intersubject variability in plasma profiles, has been an incentive for the use of excipients with absorption-enhancing properties to boost the bioavailability of poorly absorbed drugs. The development of such excipients is not a simple task, so understanding enhancement mechanisms in relation to physiology can facilitate the identification of structure-function relationships as well as the development of newer agents for customary applications. The literature is replete with reports of absorption promoters, the selection of which is influenced by the mechanisms, safety, pharmacological inertness, rapidity of action, reversibility of induced membrane alterations and excipient compatibility. Despite promising results in preliminary screenings, the development process is hindered by low reproducible efficacy and pharmacologically driven safety issues. In this review, we elaborate on the importance of permeation enhancers in oral drug delivery, their current status, and issues at the forefront of the development of formulations using absorption promoter technologies.

Liposomes and Lipid Envelope-Type Systems for Systemic siRNA Delivery.

The 'RNA interference' has emerged as a potential therapeutic strategy owing to its high specificity to silent any malfunctioned gene in diseases with genetic background. Currently intravenous delivery of siRNA has been a preferred way of administration due to high access of blood to the organs where direct delivery is not possible. Among non-viral delivery systems enabling systemic delivery of siRNA, liposomes and lipid envelope systems appear to be promising due to their biocompatibility over other systems. However, these systems are still challenged by toxicity issues, instability in blood, non-specific distribution and low transfection efficiency after intravenous administration. Therefore, to increase the success of lipid based siRNA delivery, it is essential to understand the importance of various factors affecting the efficiency of siRNA delivery. The current review focuses on the formulation of lipid based siRNA formulations, the challenges posed in systemic delivery and various aspects affecting the transfection efficiency of such formulations. The review also focuses on emerging strategies for lipid based siRNA delivery and overviews clinical prospects for better development of siRNA delivery systems in future. Considering the current trends, it seems that liposomes and lipid based envelope systems for systemic delivery of siRNA will translate into extensive clinical application overcoming the associated challenges in near future.

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.

Protein- and Peptide-drug conjugates: an emerging drug delivery technology.

Protein- and peptide-drug conjugates hold a promising stance in the delivery of therapeutic agents by providing distinct advantage of improving therapeutic potential of drugs. Recent advancements in the proteomics and recombinant DNA technology, by enabling identification of distinct structural features of proteins and making it feasible to introduce specific functionalities in protein/peptide structure, has made it possible to synthesize high quality protein- and peptide-drug conjugates though a wide variety of coupling techniques. Additionally, use of specialized linkers makes them unique in their in vivo therapeutic application by providing target tissue-specific release of drug. Several protein- and peptide-drug conjugates are currently under clinical trials warranting their huge market potential in near future. Increased understanding in this field will surely enable us to produce high quality protein- and peptide-drug conjugates which will serve therapeutic needs demanded from drug delivery systems in clinical settings.

Polymer assisted entrapment of netilmicin in PLGA nanoparticles for sustained antibacterial activity.

This study was aimed to develop poly(dl-lactide-co-glycolide) (PLGA) nanoparticle of highly water soluble antibiotic drug, netilmicin sulfate (NS) with improved entrapment efficiency (EE) and antibacterial activity. Dextran sulfate was introduced as helper polymer to form electrostatic complex with NS. Nanoparticles were prepared by double emulsification method and optimized using 2(5-1) fractional factorial design. EE was mainly influenced by dextran sulfate: NS charge ratio and PLGA concentration, whereas particle size (PS) was affected by all factors examined. The optimized NS-loaded-NPs had EE and PS of 93.23 ± 2.7% and 140.83 ± 2.4 nm respectively. NS-loaded-NPs effectively inhibited bacterial growth compared to free NS. Sustained release protected its inactivation and reduced the decline in its killing activity over time even in presence of bronchial cells. A MIC value of 18 µg/mL was observed for NPs on P. aeruginosa. Therefore, NPs with sustained bactericidal efficiency against P. aeruginosa may provide therapeutic benefit in chronic pulmonary infection, like cystic fibrosis.

Protein-functionalized PLGA nanoparticles of lamotrigine for neuropathic pain management.

Lamotrigine (LTG), a sodium and calcium channel blocker, has demonstrated efficacy for the treatment of neuropathic pain in multiple, randomized, controlled trials. However, its potential clinical applications in neuropathic pain are limited due to the risk of dose-dependent severe rashes associated with high dose and prompt dose escalation. Further, the poor pharmacokinetic profile due to non-selective distribution to organs other than brain reduces the efficacy of dosage regimen. Therefore, the aim of present investigation is to develop surface-engineered LTG nanoparticles (NPs) using transferrin and lactoferrin as ligand to deliver higher amount of drug to brain and improve the biodistribution and pharmacokinetic profile of drug with prolonged duration of action and reduced accumulation in non-target organs. The LTG NPs were prepared by nanoprecipitation and optimized by factorial design for high entrapment and optimized particle size. The optimized NPs were surface functionalized by conjugating with the lactoferrin (Lf) and transferrin (Tf) as ligands. The developed NPs were characterized for different physicochemical parameters and stability. The in vivo biodistribution showed preferential targeting to brain and reduced accumulation in non-target organs over a prolonged duration of time. Finally, partial sciatic nerve injury model was used to demonstrate the increased pharmacodynamic response as antinociceptive effect. Both biodistribution and pharmacodynamic study in mice confirmed that the approach used for LTG can help to increase clinical applications of LTG due to brain targeting and reduced side effects.

Receptor-targeted drug delivery: current perspective and challenges.

Receptor-targeted drug delivery has been extensively explored for active targeting. However, the scarce clinical applications of such delivery systems highlight the implicit hurdles in development of such systems. These hurdles begin with lack of knowledge of differential expression of receptors, their accessibility and identification of newer receptors. Similarly, ligand-specific challenges range from proper choice of ligand and conjugation chemistry, to release of drug/delivery system from ligand. Finally, nanocarrier systems, which offer improved loading, biocompatibility and reduced premature degradation, also face multiple challenges. This review focuses on understanding these challenges, and means to overcome such challenges to develop efficient, targeted drug-delivery systems.

PEG - a versatile conjugating ligand for drugs and drug delivery systems.

Polyethylene glycol (PEG) conjugation is a rapidly evolving strategy to solve hurdles in therapeutic delivery and is being used as an add-on tool to the traditional drug delivery methods. Chemically, PEGylation is a term used to denote modification of therapeutic molecules by conjugation with PEG. Efforts are constantly being made to develop novel strategies for conjugation of PEG with these molecules in order to increase its current applications. These strategies are specific to the therapeutic system used and also depend on the availability of activated PEGylating agents. Therefore, a prior knowledge is essential in selecting appropriate method for PEGylation. Once achieved, a successful PEGylation can amend the pharmacokinetic and pharmacodynamic outcomes of therapeutics. Specifically, the primary interest is in their ability to decrease uptake by reticuloendothelial system, prolong blood residence, decrease degradation by metabolic enzymes and reduce protein immunogenicity. The extensive research in this field has resulted into many clinical studies. The knowledge of outcome of these studies gave a good feedback and lessons which helped researchers to redesign PEG conjugates with improved features which can increase the chance of hitting the market. In light of this, the current paper highlights the approaches, novel strategies and the utilization of modern concept for PEG conjugation with respect to various bioactive components of clinical relevance. Moreover, this review also discusses potential clinical outcomes of the PEG conjugation, regulatory approved PEGylated product, clinical trials for newer formulations, and also provides future prospects of this technology.

Epidermal growth factor receptor targeting in cancer: a review of trends and strategies.

The epidermal growth factor receptor (EGFR) is a cell-surface receptor belonging to ErbB family of tyrosine kinase and it plays a vital role in the regulation of cell proliferation, survival and differentiation. However; EGFR is aberrantly activated by various mechanisms like receptor overexpression, mutation, ligand-dependent receptor dimerization, ligand-independent activation and is associated with development of variety of tumors. Therefore, specific EGFR inhibition is one of the key targets for cancer therapy. Two major approaches have been developed and demonstrated benefits in clinical trials for targeting EGFR; monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs). EGFR inhibitors like, cetuximab, panitumumab, etc. (mAbs) and gefitinib, erlotinib, lapatinib, etc. (TKIs) are now commercially available for treatment of variety of cancers. Recently, many other agents like peptides, nanobodies, affibodies and antisense oligonucleotide have also shown better efficacy in targeting and inhibiting EGFR. Now a days, efforts are being focused to identify molecular markers that can predict patients more likely to respond to anti-EGFR therapy; to find out combinatorial approaches with EGFR inhibitors and to bring new therapeutic agents with clinical efficacy. In this review we have outlined the role of EGFR in cancer, different types of EGFR inhibitors, preclinical and clinical status of EGFR inhibitors as well as summarized the recent efforts made in the field of molecular EGFR targeting.