Crosslinked-hybrid nanoparticle embedded in thermogel for sustained co-delivery to inner ear.

Treatment-induced ototoxicity and accompanying hearing loss are a great concern associated with chemotherapeutic or antibiotic drug regimens. Thus, prophylactic cure or early treatment is desirable by local delivery to the inner ear. In this study, we examined a novel way of intratympanically delivered sustained nanoformulation by using crosslinked hybrid nanoparticle (cHy-NPs) in a thermoresponsive hydrogel i.e. thermogel that can potentially provide a safe and effective treatment towards the treatment-induced or drug-induced ototoxicity. The prophylactic treatment of the ototoxicity can be achieved by using two therapeutic molecules, Flunarizine (FL: T-type calcium channel blocker) and Honokiol (HK: antioxidant) co-encapsulated in the same delivery system. Here we investigated, FL and HK as cytoprotective molecules against cisplatin-induced toxic effects in the House Ear Institute - Organ of Corti 1 (HEI-OC1) cells and in vivo assessments on the neuromast hair cell protection in the zebrafish lateral line. We observed that cytotoxic protective effect can be enhanced by using FL and HK in combination and developing a robust drug delivery formulation. Therefore, FL-and HK-loaded crosslinked hybrid nanoparticles (FL-cHy-NPs and HK-cHy-NPs) were synthesized using a quality-by-design approach (QbD) in which design of experiment-central composite design (DoE-CCD) following the standard least-square model was used for nanoformulation optimization. The physicochemical characterization of FL and HK loaded-NPs suggested the successful synthesis of spherical NPs with polydispersity index < 0.3, drugs encapsulation (> 75%), drugs loading (~ 10%), stability (> 2 months) in the neutral solution, and appropriate cryoprotectant selection. We assessed caspase 3/7 apopototic pathway in vitro that showed significantly reduced signals of caspase 3/7 activation after the FL-cHy-NPs and HK-cHy-NPs (alone or in combination) compared to the CisPt. The final formulation i.e. crosslinked-hybrid-nanoparticle-embedded-in-thermogel was developed by incorporating drug-loaded cHy-NPs in poloxamer-407, poloxamer-188, and carbomer-940-based hydrogel. A combination of artificial intelligence (AI)-based qualitative and quantitative image analysis determined the particle size and distribution throughout the visible segment. The developed formulation was able to release the FL and HK for at least a month. Overall, a highly stable nanoformulation was successfully developed for combating treatment-induced or drug-induced ototoxicity via local administration to the inner ear.

In vivo measurement of trabecular meshwork stiffness in a corticosteroid-induced ocular hypertensive mouse model.

Ocular corticosteroids are commonly used clinically. Unfortunately, their administration frequently leads to ocular hypertension, i.e., elevated intraocular pressure (IOP), which, in turn, can progress to a form of glaucoma known as steroid-induced glaucoma. The pathophysiology of this condition is poorly understood yet shares similarities with the most common form of glaucoma. Using nanotechnology, we created a mouse model of corticosteroid-induced ocular hypertension. This model functionally and morphologically resembles human ocular hypertension, having titratable, robust, and sustained IOPs caused by increased resistance to aqueous humor outflow. Using this model, we then interrogated the biomechanical properties of the trabecular meshwork (TM), including the inner wall of Schlemm's canal (SC), tissues known to strongly influence IOP and to be altered in other forms of glaucoma. Specifically, using spectral domain optical coherence tomography, we observed that SC in corticosteroid-treated mice was more resistant to collapse at elevated IOPs, reflecting increased TM stiffness determined by inverse finite element modeling. Our noninvasive approach to monitoring TM stiffness in vivo is applicable to other forms of glaucoma and has significant potential to monitor TM function and thus positively affect the clinical care of glaucoma, the leading cause of irreversible blindness worldwide.

Topical Formulation of Self-Assembled Antiviral Prodrug Nanomicelles for Targeted Retinal Delivery.

Topical drug administration for back of the eye delivery is extremely challenging due to the presence of protection mechanisms and physiological barriers. Self-assembled polymeric nanomicelles have emerged as promising vehicles for drug delivery. Apart from serving as an inert nanocarrier for therapeutic agents, polymeric nanomicelles are known to bypass mononuclear phagocytic system (MPS) and efflux transporters thereby improving drug bioavailability. In this investigation, a highly efficacious biotinylated lipid prodrug of cyclic cidofovir (B-C12-cCDF) was formulated within polymeric nanomicelles as a carrier for targeted retinal delivery. Polymeric nanomicelles were prepared from polyoxyethylene hydrogenated castor oil 40 (HCO-40) and octoxynol 40 (OC-40). In vitro release studies revealed that B-C12-cCDF-loaded nanomicelles released B-C12-cCDF at a faster rate in stimulated tear fluid (STF) in comparison to PBST. MTT and LDH assays demonstrated negligible cytotoxicity of B-C12-cCDF-loaded nanomicelles relative to CDF and B-C12-cCDF in HRPE (human retinal pigment epithelial, D407), HCE-T (human corneal epithelial), and CCL 20.2 (human conjunctival epithelial) cells. Confocal laser scanning microscopy and flow cytometry analyses indicated that B-C12-cCDF-loaded nanomicelles were efficiently internalized into D407 and HCE-T cells in contrast to CDF and B-C12-cCDF. Moreover, little B-C12-cCDF was also observed in the nuclei after 24 h of incubation. Polymeric nanomicelles carrying the transporter targeted prodrug did not produce any cytotoxic effects and were internalized into the cells effectively. Permeability experiments across HCE-T cells further confirmed significant transport of prodrug loaded nanomicelles and their subsequent uptake into D407 cells. These findings indicate that HCO-40/OC-40 based polymeric nanomicelles could become a promising topical delivery system for ocular administration of antiviral agents.

Composite Nanoformulation Therapeutics for Long-Term Ocular Delivery of Macromolecules.

The purpose of this investigation is to design and synthesize novel pentablock (PB) copolymer (PB-1: PCL-PLA-PEG-PLA-PCL) based nanoformulations suspended in a thermosensitive gelling copolymer (PB-2: mPEG-PCL-PLA-PCL-PEGm) termed as composite nanoformulation. The composite nanoformulation was prepared to provide a sustained delivery of macromolecules over a longer duration with negligible burst release effect. The delivery system was designed to be utilized for the treatment of posterior segment ocular diseases such as age-related (wet) macular degeneration, diabetic retinopathy, and diabetic macular edema. The novel PB copolymers were characterized for their functional groups by Fourier transform infrared spectroscopy, molecular weight and purity by (1)H NMR spectroscopy, and gel permeation chromatography. X-ray diffraction analysis was used to determine the crystallinity of copolymers. The size distribution of PB-1 nanoparticles (NPs) prepared using emulsification-solvent evaporation method was found to be ∼150 nm analyzed by nanoparticle tracking analysis. The % encapsulation efficiency and % drug loading were found to be 66.64% w/w ± 1.75 and 18.17% w/w ± 0.39, respectively, (n = 3). Different weight percentages (15 and 20 wt %) of the PB-2 copolymer have been utilized for in vitro release studies of IgG-Fab from composite nanoformulation. A negligible burst release with continuous near zero-order release has been observed from the composite nanoformulation analyzed up to 80 days. In vitro cell viability and biocompatibility studies performed on ocular (human corneal epithelial and retinal pigment epithelium) and mouse macrophage (RAW 264.7) cell lines showed that the synthesized PB copolymer based composite nanoformulations were safe for clinical applications. On the basis of the results observed, it is concluded that PB copolymer based composite nanoformulations can serve as a platform for ocular delivery of therapeutic proteins. In addition, the composite nanoformulation may provide minimal side effects associated with frequent intravitreal injections.

Optimization of novel pentablock copolymer based composite formulation for sustained delivery of peptide/protein in the treatment of ocular diseases.

This manuscript is focussed on the development of pentablock (PB) copolymer based sustained release formulation for the treatment of posterior segment ocular diseases. We have successfully synthesised biodegradable and biocompatible PB copolymers for the preparation of nanoparticles (NPs) and thermosensitive gel. Achieving high drug loading with hydrophilic biotherapeutics (peptides/proteins) is a challenging task. Moreover, small intravitreal injection volume (≤100 µL) requires high loading to develop a long term (six months) sustained release formulation. We have successfully investigated various formulation parameters to achieve maximum peptide/protein (octreotide, insulin, lysozyme, IgG-Fab, IgG, and catalase) loading in PB NPs. Improvement in drug loading can facilitate delivery of larger doses of therapeutic proteins via limited injection volume. A composite formulation comprised of NPs in gel system exhibited sustained release (without burst effect) of peptides and proteins, may serve as a platform technology for the treatment of posterior segment ocular diseases.

Nanoparticle-Based Topical Ophthalmic Gel Formulation for Sustained Release of Hydrocortisone Butyrate.

This study was conducted to develop formulations of hydrocortisone butyrate (HB)-loaded poly(D,L-lactic-co-glycolic acid) nanoparticles (PLGA NP) suspended in thermosensitive gel to improve ocular bioavailability of HB for the treatment of bacterial corneal keratitis. PLGA NP with different surfactants such as polyvinyl alcohol (PVA), pluronic F-108, and chitosan were prepared using oil-in-water (O/W) emulsion evaporation technique. NP were characterized with respect to particle size, entrapment efficiency, polydispersity, drug loading, surface morphology, zeta potential, and crystallinity. In vitro release of HB from NP showed a biphasic release pattern with an initial burst phase followed by a sustained phase. Such burst effect was completely eliminated when nanoparticles were suspended in thermosensitive gels and zero-order release kinetics was observed. In HCEC cell line, chitosan-emulsified NP showed the highest cellular uptake efficiency over PVA- and pluronic-emulsified NP (59.09 ± 6.21%, 55.74 ± 6.26%, and 62.54 ± 3.30%, respectively) after 4 h. However, chitosan-emulsified NP indicated significant cytotoxicity of 200 and 500 µg/mL after 48 h, while PVA- and pluronic-emulsified NP exhibited no significant cytotoxicity. PLGA NP dispersed in thermosensitive gels can be considered as a promising drug delivery system for the treatment of anterior eye diseases.

Emerging Trends and Translational Challenges in Drug and Vaccine Delivery.

Drug and vaccine delivery have received considerable attention in recent years [...].

Dual stimuli-responsive and sustained drug delivery NanoSensoGel formulation for prevention of cisplatin-induced ototoxicity.

Cisplatin (CisPt)-induced ototoxicity (CIO) is delineated as a consequence of CisPt-induced intracellular generation of reactive oxygen species (ROS) which can be circumvented by Bucillamine (BUC; an antioxidant drug with sulfhydryl groups) and Diltiazem (DLT, L-type calcium channel blocker). However, its effective accumulation in the Organ of Corti and cell cytoplasm is desired. Therefore, a biocompatible BUC- and DLT-nanoparticles (NPs)-impregnated dual stimuli-responsive formulation (NanoSensoGel) presented here with ROS- and thermo-responsive properties for the sustained and receptive delivery of drugs. The ROS-responsive polypropylene sulfide- methyl polyethylene glycol-2000 (PPS-mPEG2000) polymer was rationally designed, synthesized, and characterized to fabricate BUC- and DLT-loaded PPS-mPEG2000-NPs (BUC- and DLT-NPs). The fabricated BUC- and DLT-NPs showed efficient cellular uptake, intracellular delivery, ROS responsiveness, and cytoprotective effect which was characterized using cellular internalization, intracellular ROS, mitochondrial superoxide, and Caspase 3/7 assays on the House Ear Institute-Organ of Corti-1 (HEI-OC1) cells. The composite NanoSensoGel (i.e., ROS-responsive BUC- and DLT-NPs suspended in the thermo-responsive hydrogel) present in a sol state at room temperature and turned to gel above 33°C, which could be essential for retaining the formulation at the target site for long-term release. The NanoSensoGel showed sustained release of BUC and DLT following Fickian release diffusion kinetics. Overall, a novel NanoSensoGel formulation developed in this study has demonstrated its great potential in delivering therapeutics in the inner ear for prophylactic treatment of CIO, and associated hearing loss.

Novel Approaches for Overcoming Biological Barriers.

The human body poses a spectrum of biological mechanisms operating at different levels that are important for its normal functioning and development [...].

Polyphenols against infectious diseases: Controlled release nano-formulations.

The emergence of multi-drug resistant (MDR) pathogens has become a global threat and a cause of significant morbidity and mortality around the world. Natural products have been used as a promising approach to counter the infectious diseases associated with these pathogens. The application of natural products and their derivatives especially polyphenolic compounds as antibacterial agents is an active area of research, and prior studies have successfully treated a variety of bacterial infections using these polyphenolic compounds. However, delivery of polyphenolic compounds has been challenging due to their physicochemical properties and often poor aqueous solubility. In this regard, nanotechnology-based novel drug delivery systems offer many advantages, including improving bioavailability and the controlled release of polyphenolic compounds. This review summarizes the pharmacological mechanism and use of nano-formulations in developing controlled release delivery systems of naturally occurring polyphenols in infectious diseases.

Intelligent micro-/nanorobots as drug and cell carrier devices for biomedical therapeutic advancement: Promising development opportunities and translational challenges.

Nanotechnology and microfabrication approaches are playing instrumental roles in the development of innovative technologies to fight human diseases. Because of promising in vitro and preclinical outcomes, micro-/nanorobots (MNRs), are increasingly being considered for personalized and precision therapeutic diagnoses, sensing, drug delivery, and surgery. Today, designing MNR-based devices to improve the safety and efficacy of drugs for targeted cells and tissues represents a novel and promising area of therapeutic development. Progress has primarily been due to many scientific breakthroughs made in design, fabrication, and operational technologies, which greatly enhanced the capabilities of MNRs to meet the requirements of biomedical applications. This review focuses on the development and emerging biomedical applications of micro-/nanostructures encompassing nanoswimmers, nanoengines, 3D-motion nanomachines, and biologically inspired microbots, nanofish, nanorockets, etc. Promising applications of these novel devices in various therapeutic areas are discussed. We examine the impacts of the rapid progress made in developing these novel devices for drug delivery applications. We also summarize the current fabrication, scale-up development and clinical translational challenges and the main roadblocks that need to be overcome, particularly those related to patient safety and personalized medicine approaches, areas that require the design of safe innovative materials. As MNRs are new, scientists should systematically investigate their behavior, functionality, biocompatibility, toxicity, biodistribution, and efficacy before considering any potential clinical evaluations, while also ensuing that they comply with ethical principles. Although still an emerging area, MNRs are steadily becoming a realistic prospect as vital future therapeutic tools for a vast array of biomedical applications.

Extracellular Vesicles As Nanomedicine: Hopes And Hurdles In Clinical Translation.

The clinical development of cell therapies is revealing that extracellular vesicles (EVs) may become very instrumental as subcellular therapeutic adjuncts in human medicine. EVs are released by various types of cells, grown in culture, such as mesenchymal stromal cells, or obtained from patients or allogeneic donors. Some EV populations (especially species of exosomes and shed microvesicles) exhibit inherent roles in cell-cell communication, thanks to their ca. 30~1000-nm nanosize and the physiological expression of cell-specific markers on their lipid bilayer membranes. Biomedical engineers are now attempting to exploit this cellular crosstalk capacity to use EVs as smart drug delivery systems that display substantial benefits in targeting, safety, and pharmacokinetics compared to synthetic nanocarriers. In parallel, the development of a set of nano-instrumentation, biochemical tools, and preclinical assays needed for optimal characterization of both naïve and drug-loaded EVs is ongoing. Although many hurdles remain, owing to the complexity of EV populations, translation of this "subcellular therapy" platform into reality is at hand and may soon change the landscape of the therapeutic arsenal in place to treat human degenerative and metabolic pathologies as well as diseases like cancer. This article provides objective opinions, balanced between unrealistic hopes of the capacity of EVs to resolve multiple clinical issues and concrete hurdles that have to be overcome to ensure that EVs are not lost in the translation phase, so that EVs can fulfill their promise by becoming a reliable therapeutic modality.

Nanoformulation properties, characterization, and behavior in complex biological matrices: Challenges and opportunities for brain-targeted drug delivery applications and enhanced translational potential.

Nanocarriers (synthetic/cell-based have attracted enormous interest for various therapeutic indications, including neurodegenerative disorders. A broader understanding of the impact of nanomedicines design is now required to enhance their translational potential. Nanoformulations in vivo journey is significantly affected by their physicochemical properties including the size, shape, hydrophobicity, elasticity, and surface charge/chemistry/morphology, which play a role as an interface with the biological environment. Understanding protein corona formation is crucial in characterizing nanocarriers and evaluating their interactions with biological systems. In this review, the types and properties of the brain-targeted nanocarriers are discussed. The biological factors and nanocarriers properties affecting their in vivo behavior are elaborated. The compositional description of cell culture and biological matrices, including proteins potentially relevant to protein corona built-up on nanoformulation especially for brain administration, is provided. Analytical techniques of characterizing nanocarriers in complex matrices, their advantages, limitations, and implementation challenges in industrial GMP environment are discussed. The uses of orthogonal complementary characterization approaches of nanocarriers are also covered.

Extracellular Microvesicles as New Industrial Therapeutic Frontiers.

Microvesicles (MVs) are subcellular physiological vehicles present in all body fluids that mediate the transfer of intercellular information within biological systems and contribute to healthy conditions. MVs have lipid bilayer membranes decorated with multiple ligands that can interact with receptors on target cells, rendering them as promising candidates for targeted delivery. The biotechnology and cell therapy industries are developing MV-based preparations that use this subcellular therapeutic machinery (in a naïve or modified state) for regenerative medicine, as substitutes for intact cell therapy, and as intelligent targeted drug delivery carriers. However, significant production challenges must be overcome before MVs scale-up development, clinical translation, and routine therapeutic application can be realized. The unique expertise developed in the biotechnology industry should facilitate market access to MV-based therapeutics. In this review, the roles of biotechnology and cell therapy industries to manufacture MVs as inherent therapeutic agents or drug delivery systems are summarized. The manufacturing, development, characterization, and regulatory challenges for successful translation are discussed.

Facilitating the translation of nanomedicines to a clinical product: challenges and opportunities.

There are numerous hurdles hindering the clinical translation of nanomedicines. The major challenges are: reproducible manufacturing and scale-up, availability of appropriate characterization methods, instability under in vivo environments, safety issues, poor understanding of the disease heterogeneity and patient preselection strategies, regulatory barriers and inadequate understanding of the biophysical and chemical interactions of nanoformulations. Thus, a better understanding of key physicochemical attributes and their characterization methods, in vivo behavior and the in-vitro-in-vivo characterization cascade of stability, safety and efficacy testing is needed to accelerate nanomedicine translation. Technologies such as quality-by-design, process analytical techniques and microfluidics could significantly accelerate the translation of nanomedicines. However, these approaches require further learning and an adequate regulatory background. Overall, to achieve an efficient clinical translation, collaboration among academia, industry and regulatory bodies is required to ensure safe and effective nanomedicine products. This review discusses the challenges and opportunities to facilitate the translation of nanomedicines to a commercial product.

Advances and applications of block-copolymer-based nanoformulations.

Advances in polymer synthetic approaches have significantly enhanced the ability to rationally design the block copolymers with tailor-made functionality and variable molecular weight. Hence, block copolymers have been extensively applied in the formulation of nanostructure materials. Owing to their amphiphilic characteristics, block copolymers can generate different nanostructures, providing easy adjustability of their size, stability and surface chemistry. In this review, block copolymer classification, synthesis, characterization, stimuli-responsive behavior and nanostructure applications are summarized. Although block copolymers hold great potential for improving the therapeutic efficacy of drugs, a comprehensive delivery potential of these systems has not been fully exploited. Thus, an outlook on future developments on block-copolymer-based assemblies are further discussed. The new developments in block copolymers are expected to contribute significantly to the field of polymeric nanomedicine.

Ocular delivery of proteins and peptides: Challenges and novel formulation approaches.

The impact of proteins and peptides on the treatment of various conditions including ocular diseases over the past few decades has been advanced by substantial breakthroughs in structural biochemistry, genetic engineering, formulation and delivery approaches. Formulation and delivery of proteins and peptides, such as monoclonal antibodies, aptamers, recombinant proteins and peptides to ocular tissues poses significant challenges owing to their large size, poor permeation and susceptibility to degradation. A wide range of advanced drug delivery systems including polymeric controlled release systems, cell-based delivery and nanowafers are being exploited to overcome the challenges of frequent administration to ocular tissues. The next generation systems integrated with new delivery technologies are anticipated to generate improved efficacy and safety through the expansion of the therapeutic target space. This review will highlight recent advances in formulation and delivery strategies of protein and peptide based biopharmaceuticals. We will also describe the current state of proteins and peptides based ocular therapy and future therapeutic opportunities.

Nanoparticles in thermosensitive gel based composite nanosystem for ocular diseases.

The pentablock (PB) copolymers based composite nanosystems were designed to provide a long-term delivery of macromolecules to the back of the eye. A unique arrangement of each block (polyethylene glycol, polylactic acid, and polycaprolactone) with various molecular weights (PB-A and PB-B) was selected for the synthesis of nanoparticles (NPs) and thermosensitive gel (PB-C) by sequential ring-opening bulk copolymerization reaction. PB copolymers were characterized for their molecular weight and purity by 1H-NMR spectroscopy and crystallinity by PXRD. The macromolecule model drugs [lysozyme (Lyz ~ 14.5 kDa), IgG-Fab (~ 50 kDa), and IgG (~ 150 kDa)] were selected to delineate the effect of molecular weights on in vitro release profile of nanoformulations. Lyz-, Fab-, and IgG-encapsulated NPs were prepared by double emulsion solvent evaporation method. The entrapment efficiency (EE%) and drug loading (DL%) of macromolecules was higher for PB-B copolymers due to its higher molecular weight and hydrophobicity compare to PB-A. The particle size range of NPs was ~ 200-270 nm. In vitro release profiles of Lyz-, Fab-, and IgG-encapsulated in NPs alone and NPs suspended in gel (composite nanosystem) demonstrated a minimal burst release and drug release over a long period. The effect of hydrodynamic diameter of macromolecules and hydrophobicity of PB copolymers was investigated on the release profile of nanosystems. In vitro biocompatibility study showed negligible cytokine (IL-1, IL-6, and TNF-α) release, which confirmed the safety of the PB copolymers. Based on the results, it is anticipated that long-term ocular delivery of macromolecules can be achieved through composite nanosystems.

Transporter effects on cell permeability in drug delivery.

INTRODUCTION: The role of drug transporters as one of the determinants of cellular drug permeability has become increasingly evident. Despite the lipophilicity of a drug molecule as rate-limiting factor for passive diffusion across biological membranes, carrier-mediated and active transport have gained attention over the years. A better understanding of the effects and roles of these influx transporters towards transmembrane permeability of a drug molecule need to be delineated for drug development and delivery. Areas covered: This review focuses on findings relative to role of transporters in drug absorption and bioavailability. Particularly the areas demanding further research have been emphasized. This review will also highlight various transporters expressed on vital organs and their effects on drug pharmacokinetics. Expert opinion: Significant efforts have been devoted to understand the role of transporters, their iterative interplay with metabolizing enzymes through molecular enzymology, binding and structure-activity relationship studies. A few assays such as parallel artificial membrane permeation assay (PAMPA) have been developed to analyze drug transport across phospholipid membranes. Although large web-accessible databases on tissue selective expression profiles at transcriptomic as well as proteomic are available, there is a need to collocate the scattered literature on the role of transporters in drug development and delivery.