Dipesh Baradia

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.

Proteins: emerging carrier for delivery of cancer therapeutics.

Introduction: Over the past decades, proteins have emerged as versatile carriers for the diagnosis and treatment of cancer, diabetes, rheumatoid arthritis, and many more diseases. Proteins have gained considerable attention in formulation of several delivery systems for anticancer drugs due to their nontoxic, non-immunogenic, biocompatible and biodegradable nature. Proteins are good candidates for conjugation with drugs as they provide good pharmacokinetics as well as better cancer tissue accumulation. Protein nanoparticulate systems are also of advancing importance owing to their modifiable functionalities and potential applications in various biological fields. The customizable nature of proteins also makes them outstanding carriers as target-specific delivery systems. Areas covered: This review emphasizes on protein conjugates (drug-albumin, drug-gelatin, drug-transferrin, and drug-antibody conjugates), protein nanoparticles (prepared using albumin, gelatin, casein, silk proteins, elastin, and lectins), surface modification of protein nanoparticles (using surfactant, polyethylene glycol, cationic/thermosensitive polymers, folic acid, monoclonal antibodies, and peptides/proteins), and their preclinical and clinical status with respect to cancer therapy. Expert opinion: The major obstacles for commercial success of protein-based delivery are lack of inexpensive as well as quality methods for their preparation and quality control; and if overcome, proteins will stand out as a superior drug-delivery carrier for cancer therapy.

cRGD grafted liposomes containing inorganic nano-precipitate complexed siRNA for intracellular delivery in cancer cells

Development of effective vector for intracellular delivery of siRNA has always been a challenge due to its hydrophilicity, net negative surface charge and sensitivity against nucleases in biological milieu. The present investigation was aimed to develop a novel non-viral liposomal carrier for siRNA delivery. Nano-precipitate of calcium phosphate was entrapped in liposomes composed of a neutral lipid (DPPC), a fusogenic lipid (DOPE), a PEGylated lipid (DSPE-mPEG2000) and cholesterol. siRNA was made permeable through liposomal bilayer and complexed to calcium phosphate precipitates inside the liposomes. siRNA entrapped liposomes were further grafted with cRGD to achieve targeting potential against cancer cells. More than 80% of siRNA was entrapped inside the liposomes having average particle size below 150nm. Cryo-transmission electron microscopy revealed the intra-liposomal calcium phosphate precipitation and unilamellar morphology of prepared liposomes. The viability of A549 lung cancer cells was significantly higher after treatment with siRNA entrapped liposomes as compared to Lipofectamine2000 complexed siRNA. Fluorescent intensity in lung carcinoma cells was significantly higher after exposure to fluorescent siRNA entrapped liposomes than with Lipofectamine2000, which were confirmed by both confocal microscopy and flow cytometry. Live imaging by confocal microscopy ascertained the targeting efficacy of cRGD grafted liposomes compared to naked siRNA and non-grafted liposomes. Developed liposomal formulation showed effective protection of siRNA against serum nucleases along with less haemolytic potential and excellent stability against electrolyte induced flocculation. At 5nM concentration gene expression of target protein was reduced up to 24.1±3.4% while Lipofectamine2000 reduced expression level up to 26.35±1.55%. In vivo toxicity in mice suggested admirable safety profile for developed lipid based delivery vector. These results advocate that prepared liposomal system would be of high value for intracellular delivery of siRNA.

Development and Characterization of siRNA Lipoplexes: Effect of Different Lipids, In Vitro Evaluation in Cancerous Cell Lines and In Vivo Toxicity Study

ABSTRACTCationic liposomes have long been used as non-viral vectors for small interfering RNA (siRNA) delivery but are associated with high toxicity, less transfection efficiency, and in vivo instability. In this investigation, we have developed siRNA targeted to RRM1 that is responsible for development of resistance to gemcitabine in cancer cells. Effect of different lipid compositions has been evaluated on formation of stable and less toxic lipoplexes. Optimized cationic lipoplex (D2CH) system was comprised of dioleoyl-trimethylammoniumpropane (DOTAP), dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), hydrogenated soya phosphocholine (HSPC), cholesterol, and methoxy(polyethyleneglycol)2000–1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG2000–DSPE). D2CH lipoplexes have shown particle size (147.5 ± 2.89 nm) and zeta potential (12.26 ± 0.54 mV) characteristics essential for their in vivo use. In vitro cytotoxicity study has shown low toxicity of developed lipoplexes as compared with lipofectamine-2000 up to N/P ratio as high as 7.5. Cell uptake studies and gene expression studies have confirmed intracellular availability of siRNA. In addition, developed lipoplexes also showed ~3 times less hemolytic potential as compared with DOTAP/DOPE lipoplexes at lipid concentration of 5 mg/mL. Lipoplexes also maintained particle size less than 200 nm on exposure to high electrolyte concentration and showed >70% siRNA retention in presence of serum showing siRNA protection conferred by lipoplexes. Furthermore, in vivo acute toxicity studies in mice showed that formulation was non-toxic up to a dosage of 0.75 mg of siRNA/kg as lipoplexes and 300 mg lipid/kg as blank liposomes indicating tolerability of lipoplexes at a dose much higher than required for therapeutic use. Promising results of this study warrant further investigation of developed siRNA lipoplexes for cancer treatment.

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

Abstract 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 25-1 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.

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.

Brain targeted intranasal delivery of tramadol: comparative study of microemulsion and nanoemulsion

Abstract Objective: The present investigation was aimed to develop and compare microemulsion and nanoemulsion for brain targeted intranasal delivery of tramadol to achieve maximum therapeutic efficacy in treatment of episodic and emergency pain. Methods: Tramadol microemulsion (TME) and tramadol nanoemulsion (TNE) were developed and evaluated for physical properties. Ex vivo diffusion and nasal toxicity of TME and TNE were assessed by using sheep nasal mucosa. Biodistribution, pharmacokinetic and pharmacodynamic studies in mice were also performed. Results: Globule sizes of TME and TNE were 16.69 ± 3.21 and 136.3 ± 4.3 nm, respectively. TNE was found be safe with respect to multiple dosing via nasal route. Both TME and TNE were stable during accelerated stability studies. AUC(0→24) in mice brain for TME and TNE was significantly higher as compared with tramadol solution. TME and TNE displayed significantly higher antinociceptive effect for a period of 16 h as compared with tramadol solution. Discussion: TME and TNE were delivered to brain, circumventing BBB in brisk manner, establishing immediately the minimum effective concentration required for therapeutic response. Significant enhancement in antinociceptive effect was observed after intranasal delivery of TME and TNE. Conclusion: Intranasal administration of TME and TNE would be effective in management of episodic and emergency pain treatment.

Development of voriconazole loaded large porous particles for inhalation delivery: effect of surface forces on aerosolisation performance, assessment of in vitro safety potential and uptake by macrophages

The present study investigated the development of a dry powder inhalable formulation of voriconazole (VRZ) using quality by design (QbD) principles and assessment of its suitability for administration in the lungs. VRZ loaded large porous particles (VLPP) were extensively optimised using I-optimal design investigating the four factors (polymer type, porogen concentration, drug loading and homogenisation speed) at three different levels. Formulations were evaluated for desirable critical quality attributes (CQAs) such as particle size, drug entrapment efficiency, aerodynamic performance, porosity, surface energy, in vitro drug release, macrophage uptake and safety. A design space satisfying all CQAs was identified only in the case of VLPP fabricated from poly-lactide polymer (PLA). Statistical analyses suggest that all the factors and their higher order interactions influenced the morphology and physical properties of VLPP. Optimised VLPP exhibited a VRZ loading of 4.85 ± 0.39%, porosity of 0.17 ± 0.02 and median volume diameter of 8.84 ± 0.12 μm, measured with laser diffraction. Moreover, their mass median aerodynamic diameter (2.85 ± 0.38 μm) and fine particle fraction (FPF) (27.3 ± 2.7%), as measured by an 8-stage Anderson Cascade Impactor, were suitable for pulmonary delivery. VLPP was found to sustain the release of VRZ for over 7 days. Increase in the surface energy of VLPP promoted enhanced aerosolisation. No cytotoxic and inflammatory (IL-8) effect was observed when A549 cells were incubated with VLPP. In addition, VLPP was large enough to evade macrophage uptake, thus prolonging the residence time of VLPP at the site of action. Overall, this study suggests the suitability of VLPP for targeting invasive pulmonary aspergillosis by inhalation.

Surface-modified Epirubicin-HCl liposomes and its in vitro assessment in breast cancer cell-line: MCF-7

Abstract Background: Epirubicin-HCl is highly efficient for breast cancer management at a concentration of 60–90 mg/m2. However, its application is limited due to cumulative dose-dependent cardio-toxicity. Purpose: The main aim of this study was to formulate breast cancer-targeted liposomal carrier by surface conjugation of transferrin to minimize cardio-toxicity of drug along with improved pharmacokinetic profile. Method: Liposomes were formulated by ethanol injection method using HSPC, cholesterol and DSPG and later loaded with drug by the ammonium sulfate gradient method. The formulation was characterized for physicochemical properties like size, zeta potential, entrapment efficiency, TEM; in vitro tests like electro-flocculation, hemolysis and drug release; cell line study (MCF-7 cells); in vivo studies including LD50 determination, pharmacokinetic analysis, myocardial toxicity determination and stability. Results and discussion: Optimized formulation had molar ratio of 60:30:8:2 (HSPC:Chol:DSPG:mPEG-DSPE) with entrapment efficiency ∼83%, particle size below 200 nm and zeta potential about −20 mV. In vitro studies proved non-interfering property and drug release character of formulation while cell line studies demonstrated improvement in cell uptake and thereby increased cytotoxicity of targeted formulation. The IC50 value obtained for epirubicin solution, non-targeted and targeted liposomes was 0.675, 0.532 and 0.192 µg/ml, respectively. Furthermore, in vivo tests validated safety and distribution profile of prepared formulations. Conclusion: Apt properties of prepared Epirubicin-HCl liposomal formulation warrant its clinical application in breast cancer treatment after further studies.