Development and testing of nanoparticles delivery for P7C3 small molecule using injury models.

The use of nanoparticles (NPs) has emerged as a potential tool for safe and effective drug delivery. In the present study, we developed small molecule P7C3-based NPs and tested its efficacy and toxicity along with the tissue specific aptamer-modified P7C3 NPs. The P7C3 NPs were prepared using poly (D, L-lactic-co-glycolic acid) carboxylic acid (PLGA-COOH) polymer, were conjugated with skeletal muscle-specific RNA aptamer (A01B P7C3 NPs) and characterized for its cytotoxicity, cellular uptake, and wound healing in vitro. The A01B P7C3 NPs demonstrated an encapsulation efficiency of 30.2 ± 2.6%, with the particle size 255.9 ± 4.3 nm, polydispersity index of 0.335 ± 0.05 and zeta potential of + 10.4 ± 1.8mV. The FTIR spectrum of P7C3 NPs displayed complete encapsulation of the drug in the NPs. The P7C3 NPs and A01B P7C3 NPs displayed sustained drug release in vitro for up to 6 days and qPCR analysis confirmed A01B aptamer binding to P7C3 NPs. The C2C12 cells viability assay displayed no cytotoxic effects of all 3 formulations at 48 and 72 h. In addition, the cellular uptake of A01B P7C3 NPs in C2C12 myoblasts demonstrated higher uptake. In vitro assay mimicking wound healing showed improved wound closure with P7C3 NPs. In addition, P7C3 NPs significantly decreased TNF-α induced NF-κB activity in the C2C12/NF-κB reporter cells after 24-hour treatment. The P7C3 NPs showed 3-4-fold higher efficacy compared to P7C3 solutions in both wound-closure and inflammation assays in C2C12 cells. Furthermore, the P7C3 NPs showed 3-4-fold higher efficacy in reducing the infarct size and protected mouse hearts from ex vivo ischemia-reperfusion injury. Overall, this study demonstrates the safe and effective delivery of P7C3 NPs.

Nanodelivery of Resveratrol-Loaded PLGA Nanoparticles for Age-Related Macular Degeneration.

Age-related macular degeneration, precisely neovascular form, is the leading cause of vision loss and the key treatment includes intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) agents. A method to increase local concentration of drug at posterior segment of the eye and to reduce the frequency of intravitreal injections is an unmet need. Resveratrol, a naturally occurring antioxidant and anti-inflammatory polyphenol, was loaded in PLGA polymeric nanoparticles to study their sustained release property and effectiveness in reducing expression of VEGF protein in vitro. Nanoparticles were characterized using FTIR, DSC, size, encapsulation efficiency, TEM, and in vitro drug release studies. Using MTT assay, the cytotoxicity of formulation was evaluated on ARPE-19 cells. The cellular uptake and VEGF expression levels were also evaluated in in vitro settings. The optimized formulation had a particle size of 102.7 nm with - 47.30 mV of zeta potential. Entrapment efficiency was found to be 65.21%. The cell viability results suggested compatibility of developed formulation. Cellular uptake and VEGF expression levels for the formulated nanoparticles specified that the developed formulation showed potential cellular uptake and had displayed anti-angiogenic property by inhibiting VEGF expression in vitro. The results showed successful development of resveratrol-loaded nanoparticles which may be used for neovascular AMD treatment alone or in combination with anti-VEGF agents.

The effects of repetitive transcranial magnetic stimulation in older adults with mild cognitive impairment: a protocol for a randomized, controlled three-arm trial.

BACKGROUND: Mild Cognitive Impairment (MCI) carries a high risk of progression to Alzheimer's disease (AD) dementia. Previous clinical trials testing whether cholinesterase inhibitors can slow the rate of progression from MCI to AD dementia have yielded disappointing results. However, recent studies of the effects of repetitive transcranial magnetic stimulation (rTMS) in AD have demonstrated improvements in cognitive function. Because few rTMS trials have been conducted in MCI, we designed a trial to test the short-term efficacy of rTMS in MCI. Yet, in both MCI and AD, we know little about what site of stimulation would be ideal for improving cognitive function. Therefore, two cortical sites will be investigated in this trial: (1) the dorsolateral prefrontal cortex (DLPFC), which has been well studied for treatment of major depressive disorder; and (2) the lateral parietal cortex (LPC), a novel site with connectivity to AD-relevant limbic regions. METHODS/DESIGN: In this single-site trial, we plan to enroll 99 participants with single or multi-domain amnestic MCI. We will randomize participants to one of three groups: (1) Active DLPFC rTMS; (2) Active LPC rTMS; and (3) Sham rTMS (evenly split between DLPFC and LPC locations). After completing 20 bilateral rTMS treatment sessions, participants will be followed for 6 months to test short-term efficacy and track durability of effects. The primary efficacy measure is the California Verbal Learning Test-II (CVLT-II), assessed 1 week after intervention. Secondary analyses will examine effects of rTMS on other cognitive measures, symptoms of depression, and brain function with respect to the site of stimulation. Finally, selected biomarkers will be analyzed to explore predictors of response and mechanisms of action. DISCUSSION: The primary aim of this trial is to test the short-term efficacy of rTMS in MCI. Additionally, the project will provide information on the durability of cognitive effects and potentially distinct effects of stimulating DLPFC versus LPC regions. Future efforts would be directed toward better understanding therapeutic mechanisms and optimizing rTMS for treatment of MCI. Ultimately, if rTMS can be utilized to slow the rate of progression to AD dementia, this will be a significant advancement in the field. TRIAL REGISTRATION: Clinical Trials NCT03331796. Registered 6 November 2017, https://clinicaltrials.gov/ct2/show/NCT03331796. All items from the World Health Organization Trial Registration Data Set are listed in Appendix A. PROTOCOL VERSION: This report is based on version 1, approved by the DSMB on 30 November, 2017 and amended on 14 August, 2018 and 19 September, 2019.

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.

IGF-II-Conjugated Nanocarrier for Brain-Targeted Delivery of p11 Gene for Depression.

Gene therapy involving p11 cDNA has been thought to be a futuristic approach for the effective management of depression as the existing treatment regimen presents many issues regarding late onset of action, patient withdrawal and their side effects. For the effective transfection of p11 gene intracellularly, two cationic lipids based on phospholipid DOPE conjugated to basic amino acids histidine and arginine were synthesised, used for liposome formulation and evaluated for their ability as gene delivery vectors. They were further converted using IGF-II mAb into immunoliposomes for CNS targeting and mAb conjugation to liposomes were characterised by SDS-PAGE. They were further analysed by in vitro characterisation studies that include erythrocyte aggregation study, electrolyte-induced study, heparin compatibility study and serum stability studies. SHSY5Y cells were used for conducting cytotoxicity of synthesised lipids and live imaging of cell uptake for 25 min. Finally, the brain distribution studies and western blot were carried out in animals to evaluate them for their BBB permeation ability and effects on p11 protein which is believed to be a culprit. These formulated liposomes from synthesised lipids offer a promising approach for the treatment of depression.

Synthesis, docking and anticancer activity of azo-linked hybrids of 1,3,4-thia-/oxadiazoles with cyclic imides.

A series of novel analogues based on a diazole-imide pharmacophore were synthesized by diazotizing substituted 1,3,4-thia-/oxadiazol-2-amines and subsequently coupling the resulting diazonium salts with N-substituted cyclic imides. The resulting compounds C1 to C28 were characterized by various spectral methods, viz. IR, NMR and mass spectroscopy. All the synthesized compounds were tested against two human cancer cell lines: human breast adenocarcinoma cell line MCF-7 and colorectal adenocarcinoma cell line HT-29. Among the synthesized compounds, C14 (2-(4-chloro-3-((5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)diazenyl)phenyl)-4,5,6,7-tetrahydro-1H-isoindole-1,3(2H)-dione) emerged as a potential candidate against both MCF-7 and HT-29 with [Formula: see text] values of 0.09 ± 0.02 [Formula: see text]M and 0.11 ± 0.03 [Formula: see text]M, respectively. Similarly, compound C16 displayed highest anticancer activity against MCF-7 cell line with [Formula: see text] = 0.07 ± 0.02 [Formula: see text]M. Target fishing (inverse docking) using ChemMapper server identified EGFR tyrosine and CDK2 kinases as high priority targets for this pharmacophore. Computational docking (AutoDock 4.2) was used to analyse the interactions between the target proteins and active compounds.

Targeted delivery of monoclonal antibody conjugated docetaxel loaded PLGA nanoparticles into EGFR overexpressed lung tumour cells.

The aim of this study was to develop anti-EGFR antibody conjugated poly(lactide-co-glycolide) nanoparticles (NPs) to target epidermal growth factor receptor, highly expressed on non-small cell lung cancer cells to improve cytotoxicity and site specificity. Cetuximab was conjugated to docetaxel (DTX) loaded PLGA NPs by known EDC/NHS chemistry and characterised for size, zeta potential, conjugation efficiency and the results were 128.4 ± 3.6 nm, -31.0 ± 0.8 mV, and 39.77 ± 3.4%, respectively. In vitro release study demonstrated sustained release of drug from NPs with 25% release at pH 5.5 after 48 h. In vitro cytotoxicity studies demonstrated higher anti-proliferative activity of NPs than unconjugated NPs. Cell cycle analysis and apoptosis study were performed to evaluate extent of cell arrest at different phases and apoptotic potential for the formulations, respectively. In vivo efficacy study showed significant reduction in tumour growth and so antibody conjugated NPs present a promising active targeting carrier for tumour selective therapeutic 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.

An algorithm for optimal fusion of atlases with different labeling protocols.

In this paper we present a novel label fusion algorithm suited for scenarios in which different manual delineation protocols with potentially disparate structures have been used to annotate the training scans (hereafter referred to as "atlases"). Such scenarios arise when atlases have missing structures, when they have been labeled with different levels of detail, or when they have been taken from different heterogeneous databases. The proposed algorithm can be used to automatically label a novel scan with any of the protocols from the training data. Further, it enables us to generate new labels that are not present in any delineation protocol by defining intersections on the underling labels. We first use probabilistic models of label fusion to generalize three popular label fusion techniques to the multi-protocol setting: majority voting, semi-locally weighted voting and STAPLE. Then, we identify some shortcomings of the generalized methods, namely the inability to produce meaningful posterior probabilities for the different labels (majority voting, semi-locally weighted voting) and to exploit the similarities between the atlases (all three methods). Finally, we propose a novel generative label fusion model that can overcome these drawbacks. We use the proposed method to combine four brain MRI datasets labeled with different protocols (with a total of 102 unique labeled structures) to produce segmentations of 148 brain regions. Using cross-validation, we show that the proposed algorithm outperforms the generalizations of majority voting, semi-locally weighted voting and STAPLE (mean Dice score 83%, vs. 77%, 80% and 79%, respectively). We also evaluated the proposed algorithm in an aging study, successfully reproducing some well-known results in cortical and subcortical structures.

Bayesian segmentation of brainstem structures in MRI.

In this paper we present a method to segment four brainstem structures (midbrain, pons, medulla oblongata and superior cerebellar peduncle) from 3D brain MRI scans. The segmentation method relies on a probabilistic atlas of the brainstem and its neighboring brain structures. To build the atlas, we combined a dataset of 39 scans with already existing manual delineations of the whole brainstem and a dataset of 10 scans in which the brainstem structures were manually labeled with a protocol that was specifically designed for this study. The resulting atlas can be used in a Bayesian framework to segment the brainstem structures in novel scans. Thanks to the generative nature of the scheme, the segmentation method is robust to changes in MRI contrast or acquisition hardware. Using cross validation, we show that the algorithm can segment the structures in previously unseen T1 and FLAIR scans with great accuracy (mean error under 1mm) and robustness (no failures in 383 scans including 168 AD cases). We also indirectly evaluate the algorithm with a experiment in which we study the atrophy of the brainstem in aging. The results show that, when used simultaneously, the volumes of the midbrain, pons and medulla are significantly more predictive of age than the volume of the entire brainstem, estimated as their sum. The results also demonstrate that the method can detect atrophy patterns in the brainstem structures that have been previously described in the literature. Finally, we demonstrate that the proposed algorithm is able to detect differential effects of AD on the brainstem structures. The method will be implemented as part of the popular neuroimaging package FreeSurfer.

In vitro modulator responsiveness of 655 CFTR variants found in people with cystic fibrosis.

BACKGROUND: In 2017, the US Food and Drug Administration initiated expansion of drug labels for the treatment of cystic fibrosis (CF) to include CF transmembrane conductance regulator (CFTR) gene variants based on in vitro functional studies. This study aims to identify CFTR variants that result in increased chloride (Cl-) transport function by the CFTR protein after treatment with the CFTR modulator combination elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA). These data may benefit people with CF (pwCF) who are not currently eligible for modulator therapies. METHODS: Plasmid DNA encoding 655 CFTR variants and wild-type (WT) CFTR were transfected into Fisher Rat Thyroid cells that do not natively express CFTR. After 24 h of incubation with control or TEZ and ELX, and acute addition of IVA, CFTR function was assessed using the transepithelial current clamp conductance assay. Each variant's forskolin/cAMP-induced baseline Cl- transport activity, responsiveness to IVA alone, and responsiveness to the TEZ/ELX/IVA combination were measured in three different laboratories. Western blots were conducted to evaluate CFTR protein maturation and complement the functional data. RESULTS AND CONCLUSIONS: 253 variants not currently approved for CFTR modulator therapy showed low baseline activity (<10 % of normal CFTR Cl- transport activity). For 152 of these variants, treatment with ELX/TEZ/IVA improved the Cl- transport activity by ≥10 % of normal CFTR function, which is suggestive of clinical benefit. ELX/TEZ/IVA increased CFTR function by ≥10 percentage points for an additional 140 unapproved variants with ≥10 % but <50 % of normal CFTR function at baseline. These findings significantly expand the number of rare CFTR variants for which ELX/TEZ/IVA treatment should result in clinical benefit.

Examining the use of ICD-9 diagnosis codes for primary immune deficiency diseases in New York State.

PURPOSE: To use International Classification of Disease Codes (ICD-9) codes to investigate primary immune deficiency (PID) in New York State. METHODS: We investigated the diagnosis of Primary Immune Deficiency (PID) in New York State (NYS) using the Statewide Planning and Research Cooperative System (SPARCS) database, a comprehensive data reporting system that collects ICD-9 codes for each patient hospitalized in NYS. RESULTS: From 2000-2004 there were 13,539,358 hospitalizations for 4,777,295 patients; of these, 2,361 patients (0.05 %) were diagnosed with one or more of the ICD-9 codes for PID. Antibody defects were the most common diagnoses made. The PID population had significantly more Caucasians, and fewer African American or Hispanic subjects compared to the general population. Subjects with PID codes were younger, had longer hospitalizations, were less likely to have Medicare and more likely to have Medicaid or Blue Cross insurance. Most hospitalizations were due to respiratory and infectious diseases. Most patients resided in the most populous counties, Kings, New York and Queens, but the distribution of home zip codes was not proportional to county populations. CONCLUSIONS: These data provide useful information on incidence and complications of selected PID diagnoses in one large state.

Formulation and Characterization of Raloxifene Nanostructured Lipid Carriers for Permeability and Uptake Enhancement Applications.

Raloxifene (RLX), a biopharmaceutical classification system (BCS) class II drug, is a selective estrogen receptor modulator (SERM) having an estrogenic effect on the bone and an antiestrogenic effect on the endometrium and breast. Low solubility, high permeability, high metabolism, and low bioavailability are the characteristics of raloxifene. Although 60% is absorbed orally, raloxifene shows extremely poor bioavailability (2%) owing to its low solubility and extensive (>90%) intestinal/hepatic first-pass metabolism. Hence, it becomes important to increase the solubility of raloxifene to enhance its bioavailability. In this study, raloxifene nanostructured lipid carriers (RNLCs) were prepared using the melt dispersion ultrasonication method. The prepared RNLCs were characterized, and the in vitro studies were carried out in the human epithelial breast cancer cell line (MCF-7). The RNLCs had a size of 114.8 ± 0.98 nm and a zeta potential of +9.21 ± 0.58 mV. Transmission electron microscopy (TEM) images showed particle size ranging from 65 to 120 nm. With an entrapment efficiency of 75.04% ± 2.75%, the RNLCs showed sustained release over 7 days compared with the raloxifene drug solution. The prepared RNLCs were successfully taken up by the MCF-7 cells in a time-dependent manner, and the RNLCs showed increased cell cytotoxicity compared with the raloxifene drug. Using the parallel artificial membrane permeability assay (PAMPA), the permeability rate for raloxifene solution was calculated to be 8 × 10-6 cm/s, and for the RNLCs, it was calculated to be 17.8 × 10-6 cm/s. Hence, from the permeability rate calculated, we could conclude that raloxifene, when formulated as nanostructured lipid carriers, showed increased permeability. Overall, the prepared RNLCs were found to be superior to the raloxifene drug as such.

Honokiol-Loaded Methoxy Poly (Ethylene Glycol) Polycaprolactone Micelles for the Treatment of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD), a multifactorial age-related retinal hypoxic disorder resulting in irreversible loss of vision, is the foremost cause of blindness in the United States. Current treatment strategies involve multiple intraocular injections of antivascular endothelial growth factor (VEGF) agents into the vitreous of eye. In addition to the challenges of drug localization and targeted delivery, the need of frequent injections into the eye raises patient compliance issues, and thus call for development of sustained drug delivery systems. In this study, a sustained drug delivery system was prepared by loading an antihypoxia-induced factor (HIF) agent, honokiol (HON), into methoxy poly (ethylene glycol) polycaprolactone (MPEG-PCL) polymer. These HON-MPEG-PCL micelles were characterized by evaluating size, ζ potential, in vitro drug release profile, and morphology by transmission electron microscopy. The cytotoxic nature of developed micelles was assessed on human retinal pigment epithelial cell line (ARPE-19) cells by cytotoxicity assay. The cellular uptake and HIF and VEGF expression levels were determined in in vitro settings. Micelles formed had a particle size of 30.8 ± 0.8 nm with the poly dispersity index of 0.19 ± 0.0004 and ζ potential was found to be -5.46 ± 0.49 mv. Entrapment efficiency was calculated to be 64 ± 0.135%. In vitro drug release showed sustained release of drug from the formulation. Result from in vitro cytotoxicity study confirmed noncytotoxic nature of HON-MPEG-PCL micelles compared to HON drug solution. Furthermore, enzyme-linked immunosorbent assay studies performed showed the periodic downregulation of HIF and VEGF, which are major growth factors involved in underlying mechanism of AMD. The results showed successful development of HON-MPEG-PCL micelles, which may be useful for the effective treatment of AMD.

Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

High-dimensional pattern regression using machine learning: from medical images to continuous clinical variables.

This paper presents a general methodology for high-dimensional pattern regression on medical images via machine learning techniques. Compared with pattern classification studies, pattern regression considers the problem of estimating continuous rather than categorical variables, and can be more challenging. It is also clinically important, since it can be used to estimate disease stage and predict clinical progression from images. In this work, adaptive regional feature extraction approach is used along with other common feature extraction methods, and feature selection technique is adopted to produce a small number of discriminative features for optimal regression performance. Then the Relevance Vector Machine (RVM) is used to build regression models based on selected features. To get stable regression models from limited training samples, a bagging framework is adopted to build ensemble basis regressors derived from multiple bootstrap training samples, and thus to alleviate the effects of outliers as well as facilitate the optimal model parameter selection. Finally, this regression scheme is tested on simulated data and real data via cross-validation. Experimental results demonstrate that this regression scheme achieves higher estimation accuracy and better generalizing ability than Support Vector Regression (SVR).

Nanoscale delivery systems in treatment of posterior ocular neovascularization: strategies and potential applications.

Pathologic posterior neovascularization of eye is a major cause of irreversible vision loss and limitations of therapeutics to be successfully delivered to back of the eye has been a main obstacle for its effective treatment. Current pharmacological treatment using anti-VEGF agents being delivered intravitreally are effective but complicated due to anatomical and physiological barriers, as well as administration of high and frequent doses. With expanding horizons of nanotechnology, it can be possible to formulate promising nanoscale delivery system to improve penetration and sustained the release of therapeutic in posterior segment of the eye. Taking into consideration advances in the field of nanoscale delivery systems, this special report focuses on emerging strategies and their applications for treatment of posterior ocular neovascularization.

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.

Axitinib-Loaded Poly(Lactic-Co-Glycolic Acid) Nanoparticles for Age-Related Macular Degeneration: Formulation Development and In Vitro Characterization.

Despite all the research aiming to treat ocular diseases, age-related macular degeneration (AMD) remains one of the serious diseases worldwide, which needs to be treated. Neovascularization is a key factor in AMD and thus antiangiogenic therapy is beneficial in reducing the development of new abnormal blood vessels. Axitinib, multireceptor tyrosine kinase inhibitor, is a small molecule that works by blocking vascular endothelial growth factor receptors (VEGFR) and platelet-derived growth factor receptors (PDGFR) responsible for developing neovascularization. The goal of this study is to develop a sustained release formulation of axitinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles to minimize frequent administration of the drug by intravitreal injection. The nanoparticles were characterized for particle size and zeta potential, as well as using differential scanning calorimetry, transmission electrode microscope, and in vitro drug release profile. The cytotoxicity of the formulation was evaluated on human retinal pigmented epithelium ARPE19 cells by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide salt] assay. The cellular uptake, antimigration assay, and vascular endothelial growth factor (VEGF) expression levels were found out in vitro using cells. The optimized formulation was 131.33 ± 31.20 nm in size with -4.63 ± 0.76 mV zeta potential. Entrapment efficiency was found to be 87.9% ± 2.7%. The cytotoxicity of ARPE19 cells was <12% for nanoparticles suggesting the in vitro compatibility at 10 µM concentration of drug. Cellular uptake, antimigration assay, and VEGF expression levels for the nanoparticles suggested greater uptake, significant antiangiogenic potential, and inhibition of VEGF activity. The results showed successful development of axitinib-loaded PLGA nanoparticles as an alternative potential treatment for AMD.