Next generation therapeutics for retinal neurodegenerative diseases.

Neurodegenerative diseases affecting the visual system encompass glaucoma, macular degeneration, retinopathies, and inherited genetic disorders such as retinitis pigmentosa. These ocular pathologies pose a serious burden of visual impairment and blindness worldwide. Current treatment modalities include small molecule drugs, biologics, or gene therapies, most of which are administered topically as eye drops or as injectables. However, the topical route of administration faces challenges in effectively reaching the posterior segment and achieving desired concentrations at the target site, while injections and implants risk severe complications, such as retinal detachment and endophthalmitis. This necessitates the development of innovative therapeutic strategies that can prolong drug release, deliver effective concentrations to the back of the eye with minimal systemic exposure, and improve patient compliance and safety. In this review, we introduce retinal degenerative diseases, followed by a discussion of the existing clinical standard of care. We then delve into detail about drug and gene delivery systems currently in preclinical and clinical development, including formulation and delivery advantages/drawbacks, with a special emphasis on potential for clinical translation.

Engineered peptide-drug conjugate provides sustained protection of retinal ganglion cells with topical administration in rats.

Effective eye drop delivery systems for treating diseases of the posterior segment have yet to be clinically validated. Further, adherence to eye drop regimens is often problematic due to the difficulty and inconvenience of repetitive dosing. Here, we describe a strategy for topically dosing a peptide-drug conjugate to achieve effective and sustained therapeutic sunitinib concentrations to protect retinal ganglion cells (RGCs) in a rat model of optic nerve injury. We combined two promising delivery technologies, namely, a hypotonic gel-forming eye drop delivery system, and an engineered melanin binding and cell-penetrating peptide that sustains intraocular drug residence time. We found that once daily topical dosing of HR97-SunitiGel provided up to 2 weeks of neuroprotection after the last dose, effectively doubling the therapeutic window observed with SunitiGel. For chronic ocular diseases affecting the posterior segment, the convenience of an eye drop combined with intermittent dosing frequency could result in greater patient adherence, and thus, improved disease management.

Machine learning-driven multifunctional peptide engineering for sustained ocular drug delivery.

Sustained drug delivery strategies have many potential benefits for treating a range of diseases, particularly chronic diseases that require treatment for years. For many chronic ocular diseases, patient adherence to eye drop dosing regimens and the need for frequent intraocular injections are significant barriers to effective disease management. Here, we utilize peptide engineering to impart melanin binding properties to peptide-drug conjugates to act as a sustained-release depot in the eye. We develop a super learning-based methodology to engineer multifunctional peptides that efficiently enter cells, bind to melanin, and have low cytotoxicity. When the lead multifunctional peptide (HR97) is conjugated to brimonidine, an intraocular pressure lowering drug that is prescribed for three times per day topical dosing, intraocular pressure reduction is observed for up to 18 days after a single intracameral injection in rabbits. Further, the cumulative intraocular pressure lowering effect increases ~17-fold compared to free brimonidine injection. Engineered multifunctional peptide-drug conjugates are a promising approach for providing sustained therapeutic delivery in the eye and beyond.

Nanofiber-based glaucoma drainage implant improves surgical outcomes by modulating fibroblast behavior.

Biomaterials are implanted in millions of individuals worldwide each year. Both naturally derived and synthetic biomaterials induce a foreign body reaction that often culminates in fibrotic encapsulation and reduced functional lifespan. In ophthalmology, glaucoma drainage implants (GDIs) are implanted in the eye to reduce intraocular pressure (IOP) in order to prevent glaucoma progression and vision loss. Despite recent efforts towards miniaturization and surface chemistry modification, clinically available GDIs are susceptible to high rates of fibrosis and surgical failure. Here, we describe the development of synthetic, nanofiber-based GDIs with partially degradable inner cores. We evaluated GDIs with nanofiber or smooth surfaces to investigate the effect of surface topography on implant performance. We observed in vitro that nanofiber surfaces supported fibroblast integration and quiescence, even in the presence of pro-fibrotic signals, compared to smooth surfaces. In rabbit eyes, GDIs with a nanofiber architecture were biocompatible, prevented hypotony, and provided a volumetric aqueous outflow comparable to commercially available GDIs, though with significantly reduced fibrotic encapsulation and expression of key fibrotic markers in the surrounding tissue. We propose that the physical cues provided by the surface of the nanofiber-based GDIs mimic healthy extracellular matrix structure, mitigating fibroblast activation and potentially extending functional GDI lifespan.

Chlorin e6 Conjugated Methoxy-Poly(Ethylene Glycol)-Poly(D,L-Lactide) Glutathione Sensitive Micelles for Photodynamic Therapy.

PURPOSE: In this study, we developed a polymeric micellar system for glutathione-mediated intracellular delivery of a photosensitizer, chlorin e6 (Ce6) by synthesizing an amphiphilic polymer, methoxy-poly(ethylene glycol)-poly(D,L-lactide)-disulfide-Ce6 (mPEG-PLA-S-S-Ce6), which self-assembled in aqueous environment to form micelles. METHODS: The polymer-drug conjugate was characterized by NMR. The singlet oxygen (2O1) generation and in vitro release of Ce6 micelles were evaluated. Further, glutathione-mediated intracellular drug delivery was assessed in human alveolar adenocarcinoma cells (A549), mouse mammary carcinoma cells (4 T1) and 3D A549 spheroids. RESULTS: The micellar system protected Ce6 from aggregation leading to improved 2O1 generation compared to free Ce6. Due to the availability of glutathione, the disulfide bonds in the micelles were cleaved resulting in rapid release of Ce6 evident from the in vitro study. The Ce6 micelles displayed quicker drug release in presence of glutathione monoester (GSH-OEt) pre-treated A549 and 4 T1 cells compared to without pre-treated cells. In vitro phototoxicity of micelles displayed enhanced toxicity in 10 mM GSH-OEt pre-treated A549 and 4 T1 cells compared to untreated cells. As anticipated, Ce6 micelles showed lower drug release in presence of 0.1 mM of buthionine sulfoximine (BSO) pretreated A549 and 4 T1 cells exhibiting lower phototoxicity. Further, A549 3D spheroids treated with Ce6 micelles showed significant inhibition in growth, enhanced phototoxicity, and cellular apoptosis in comparison to free Ce6. CONCLUSION: The above results showed that the developed strategy could be effective in improving the PDT efficacy of Ce6, and the developed polymeric micellar system could be utilized as a PDT regimen for cancer.

Development of chlorin e6-conjugated poly(ethylene glycol)-poly(d,l-lactide) nanoparticles for photodynamic therapy.

AIM: In this study, we developed a chlorin e6-conjugated methoxy-poly(ethylene glycol)-poly(d,l-lactide) (mPEG-PLA-Ce6) amphiphilic polymer, which self-assembled to form stable nanoparticles. MATERIALS & METHODS: The nanoparticles were characterized for particle size, ζ-potential and singlet oxygen (1O2) generation. Cellular internalization and phototoxicity were investigated against monolayer and 3D spheroids of human lung adenocarcinoma cells (A549). RESULTS & CONCLUSION: mPEG-PLA-Ce6 exhibited a size of 149.72 ± 3.51 nm and ζ-potential of -24.82 ± 2.94 mV. The 1O2 generation by mPEG-PLA-Ce6 in water was considerably higher than free chlorin e6. The nanoparticles showed enhanced cellular internalization and phototoxicity in monolayer and 3D spheroids. The developed mPEG-PLA-Ce6 has potential application as a nanocarrier of chlorin e6 for photodynamic therapy of solid tumors.

Biotin functionalized PEGylated poly(amidoamine) dendrimer conjugate for active targeting of paclitaxel in cancer.

In the current study, we employed poly(amidoamine) (PAMAM) dendrimers of generation 4 (G4) to deliver paclitaxel (PTX), a poorly soluble anti-cancer agent precisely to cancer cells via its conjugation on dendrimer surface. Further, G4 PAMAM has been PEGylated (PEG) and tagged with Biotin, an essential micronutrient for cellular functions, receptors of which are overexpressed in certain cancers. The synthesized multifunctional conjugates were characterized by 1H NMR and zeta potential analysis techniques. In addition, the conjugates were evaluated in vitro in cell monolayers and 3D spheroids of biotin receptor over-expressed A549 cell line (human non-small cell lung cancer). G4 PTX PEG-Biotin conjugate penetrated at significantly higher extent in monolayers as well as spheroids as studied by flow cytometry and confocal microscopy by visualizing the cells at varied depth. The G4 PTX PEG-Biotin conjugate demonstrated higher cytotoxicity compared to free PTX and G4 PTX PEG conjugate as assessed by MTT assay in monolayers and Presto Blue assay in detached spheroidal cells. G4 PTX PEG-Biotin demonstrated significant inhibition of growth of tumor spheroids. Therefore, the newly synthesized biotin anchored PTX-conjugated dendrimer system is promising and could be further explored for efficiently delivering PTX to biotin receptor overexpressed cancers.

Evaluation of Anti-Tumor Efficacy of Vorinostat Encapsulated Self-Assembled Polymeric Micelles in Solid Tumors.

Vorinostat (VOR), a potent HDAC inhibitor, suffers from low solubility and poor absorption, which hinders its successful application in therapy, especially in the treatment of solid tumors. In this study, an effort to improve the physicochemical characteristics of VOR was made by encapsulating it in PEG-PLGA copolymeric micelles. VOR-loaded PEG-PLGA micelles (VOR-PEG-PLGA) were produced by thin-film hydration and physicochemically characterized. The PEG-PLGA micelles had an average size of 124.06 ± 2.6 nm, polydispersity index of 0.27 ± 0.1, and entrapment efficiency of 90 ± 2.1%. Micelles were characterized by TEM, DSC, and drug release studies. The drug release occurred in a sustained manner up to 72 h from PEG-PLGA micelles. In the in vitro cell-based studies using human breast cancer (MDA MB 231) and murine melanoma (B16F10) cell lines, VOR-PEG-PLGA micelles exhibited superior cellular internalization, enhanced cytotoxic activity, and greater apoptosis compared to free drug. Percent cell killing of 54.9% for VOR-PEG-PLGA-treated cells was observed after 24 h compared to 36% for free VOR in MDA MB 231 cell line. Further, significant tumor suppression was witnessed in B16F10 tumor-bearing mice treated with VOR-PEG-PLGA micelles with a 1.78-fold reduction in tumor volume compared to free VOR-treated animals. Overall, the VOR-PEG-PLGA micelles improved the biopharmaceutical properties of VOR, which resulted in enhanced anti-tumor efficacy. Therefore, the newly developed nano-formulation of VOR could be considered as an effective treatment option in solid tumors.

Cholesterol-grafted chitosan micelles as a nanocarrier system for drug-siRNA co-delivery to the lung cancer cells.

Combined delivery of a therapeutic small interfering RNA (siRNA) and a chemotherapeutic agent to cancer cells is promising as anticancer therapy, which could offer enhanced cell killing potential and low side effect. However, simultaneous delivery to tumor is challenging. In our study, cholesterol-modified low molecular weight chitosan (MW ~ 15 kDa) was employed as a self-assembled delivery system for both siRNA and a hydrophobic chemotherapeutic agent, curcumin to cancer cells. The siRNA/curcumin loaded nanoparticles (C-CCM/siRNA) were physico-chemically characterized for particle size (165 ±â€¯2.6 nm) and zeta potential (+24.8 ±â€¯2.2 mV). The ability of CCM to condense siRNA was determined by ethidium bromide exclusion and gel retardation assay using electrophoresis. The result demonstrated that the condensation of C-CCM with siRNA was optimum at minimum N/P ratio of 40. C-CCM/siRNA was stable at 4 °C for a period of >1 month. C-CCM/siRNA was taken up efficiently by human lung carcinoma cells, A549 in a time-dependent manner. The cellular internalization of C-CCM/siRNA was observed via clathrin-mediated endocytosis as determined by using specific endocytosis inhibitors. The study demonstrated the feasibility of the use of cholesterol conjugated chitosan as a co-delivery system for both siRNA and a hydrophobic drug for combination cancer therapy.

Transferrin-anchored poly(lactide) based micelles to improve anticancer activity of curcumin in hepatic and cervical cancer cell monolayers and 3D spheroids.

In recent years, actively targeted drug delivery systems have been utilized in pre-clinical studies for site-specific delivery of drugs, which reduces toxicities associated with chemotherapy. This study reports the preparation of the tumor homing ligand, transferrin (Tf) anchored methoxy-polyethylene glycol-poly(d,l-Lactide) polymeric micelles (Tf-PP). Curcumin which possess wide anti-cancer activity was loaded into the micelles. Tf-PPC with average particle size of 132.16 ±â€¯1.37 nm and encapsulation efficiency of 88.27 ±â€¯2.53% showed a sustained drug release. The efficacy of Tf-PPC was studied in vitro in Tf-overexpressing human cervical carcinoma (HeLa) and human hepatoma (HepG2) cells. The mouse embryo fibroblast (NIH-3T3) cells were used as control cells. Tf-PPC showed higher internalization compared to non-targeted micelles (PPC). The curcumin-mediated cytotoxicity increased significantly following Tf-PPC treatment in both the tested cell lines. In NIH-3T3 cells, Tf conjugation did not differ in comparison to the non-targeted micelles. Further, the efficiency of Tf-PPC was studied in three-dimensional (3D) HeLa tumor spheroids. The Tf-PPC was efficiently internalized by the spheroidal structures, causing higher cytotoxicity and apoptosis compared to PPC. These results reveal that the newly developed, Tf-PPC could be employed as an effective chemotherapy in the treatment of Tf- overexpressing cancers.

Octa-arginine modified poly(amidoamine) dendrimers for improved delivery and cytotoxic effect of paclitaxel in cancer.

Cell penetrating peptides (CPP) have the ability to penetrate the cell membrane and have been associated with various cargos for their facile intracellular translocation. The current study involves the synthesis of a CPP, octa-arginine (R8)-modified poly(amidoamine) dendrimer of generation 4 (G4), which has additionally been PEGylated and conjugated to the poorly soluble anticancer drug, paclitaxel (PTX). The synthesized dendrimer conjugates were characterized by proton nuclear magnetic resonance (1H-NMR) Spectroscopy and zeta potential measurements and evaluated in vitro in cell monolayers and 3D spheroids. Cellular uptake study in human cervical cancer cell line (HeLa) revealed that R8 modification significantly improved the cell association of conjugates. G4-PTX- polyethylene glycol (PEG)-R8 conjugate demonstrated enhanced cytotoxic potential and higher induction of apoptosis compared to free PTX and G4-PTX-PEG. Further, the penetrability of fluorescently labeled F-G4-PTX-PEG-R8 was evaluated in 3D spheroids of HeLa at various depths by using confocal microscopy. G4-PTX-PEG-R8 induced cell death and inhibited the growth in 3D spheroids as competently as in monolayers. The enhanced intracellular translocation of R8-modified dendrimers resulted in improved anticancer efficacy of PTX. Therefore, the newly developed dendrimer system is efficient for the intracellular delivery of PTX in cancer cells and has a strong potential to be utilized as an effective chemotherapeutic agent for cancer.

Development of Curcumin-Loaded Solid Lipid Nanoparticles Utilizing Glyceryl Monostearate as Single Lipid Using QbD Approach: Characterization and Evaluation of Anticancer Activity Against Human Breast Cancer Cell Line.

BACKGROUND: Solid lipid nanoparticles (SLNs) represent an affordable, easily scalable, stable and biocompatible drug delivery system with a high drug to lipid ratio which also improves solubility of poorly soluble drugs. OBJECTIVE: SLNs were developed by using glyceryl monostearate as the single lipid in the presence of surfactant Poloxamer 188 and evaluated the efficiency of SLNs to load the therapeutic cargo, curcumin (CUR). METHOD: The nano-formulation was optimized by Quality by Design approach to understand the effect of various process parameters on various quality attributes, including drug loadability, particle size and polydispersity. The nanoparticles were characterized using Differential scanning calorimetry (DSC), Fourier Transform Infra-red Spectroscopy (FT-IR) and X-Ray Diffraction (XRD) analysis. These novel SLNs were evaluated for in-vitro anticancer activity using breast adenocarcinoma cells (MDA-MB-231). RESULTS: The optimized formulation had a particle size of 226.802±3.92 nm with low polydispersity index of 0.244±0.018. The % encapsulation of CUR into SLNs was found to be 67.88±2.08 %. DSC, FT-IR and XRD confirmed that the CUR was encapsulated stably into the lipid matrix, thereby improving the solubility of the drug. CUR-SLN showed sustained drug release in comparison to the free CUR solution. CUR-SLNs exhibited higher cellular uptake in human breast adenocarcinoma cells compared to free CUR at both 1 and 4 h time points. CUR-SLNs demonstrated decreased cell viability (43.97±1.53%) compared to free CUR (59.33±0.95%) at a concentration of 50 µg/mL after 24 h treatment. Furthermore, the treatment of MDA-MB-231 cells with CUR-SLNs for 24 h induced significantly higher apoptosis (37.28±5.3%) in cells compared to the free CUR (21.06±0.97%). CONCLUSION: The results provide a strong rationale for further exploration of the newly developed CUR-SLN to be utilized as a potent chemotherapeutic agent in cancer therapy.

Cholesterol and vitamin E-conjugated PEGylated polymeric micelles for efficient delivery and enhanced anticancer activity of curcumin: evaluation in 2D monolayers and 3D spheroids.

A newly synthesized PEGylated cholesterol/α-tocopheryl succinate (α-TOS) linked polymer (CV) was self-assembled and loaded with curcumin to form a micellar system (C-CVM). The tri-functionalized amphiphilic polymer was constituted of hydrophobic cholesterol and α-TOS connected to hydrophilic PEG via a lysine linker. The synthesized polymer and the micelles were characterized by 1H NMR, DLS, zeta potentiometer, TEM, CMC determination and hemolysis studies. CVM displayed low CMC value of 15 µM with extent of hemolysis as less than 4%. The stable C-CVM with optimum % drug loading (14.2 ± 0.24) displayed Z average of 175.8 ± 0.68 nm with PDI (0.248 ± 0.075) and released curcumin in sustained manner in the in vitro drug release study. C-CVM demonstrated dose-dependent cellular uptake and cytotoxicity in murine melanoma, B16F10 and human breast cancer, MDA-MB-231 cell lines. CV exhibited marked reversal of drug resistance as indicated by significantly higher retention of P-glycoprotein substrate, rhodamine-123 in the resistant B16F10 cell line compared to standard P-glycoprotein inhibitor, verapamil. C-CVM demonstrated significantly higher spheroidal growth inhibition compared to C-PPM. The results provide strong evidence for CVM as promising drug delivery system and confirm the potential of C-CVM as chemotherapy in cancer.

Formulation optimization, characterization, and evaluation of in vitro cytotoxic potential of curcumin loaded solid lipid nanoparticles for improved anticancer activity.

The aim of the present research was to develop a novel, biocompatible, amenable to industrial scale up and affordable solid lipid nanoparticles (SLN) preparation of curcumin and evaluate the therapeutic efficacy in vitro using cancer cells. We have incorporated cholesterol as the lipid to prepare SLN along with the Poloxamer-188 as stabilizer. High shear homogenization was used to prepare the SLN and formulation was optimized using Quality by Design The optimized Chol CUR SLN exhibited a narrow size distribution with a particle size of 166.4±3.5nm. Percentage encapsulation (%EE) was found to be 76.9±1.9%. The SLN were further characterized by DSC, FTIR, XRD and drug release. In vitro cell studies in MDA-MB-231 (Human Breast cancer) cell line revealed that the Chol CUR SLN showed superior cytotoxicity and uptake in comparison to the free curcumin. Furthermore, Chol CUR SLN induced a significantly higher apoptosis compared to free CUR treatment. These results indicated that the curcumin encapsulated in Chol SLN was able to significantly improve the cytotoxic potential and induction of apoptosis in MDA-MB-231 cells. The promising result from our study could lead a further exploration of this nanoparticle formulation to be utilized clinically for cancer treatment.

Cholesterol-conjugated poly(D, L-lactide)-based micelles as a nanocarrier system for effective delivery of curcumin in cancer therapy.

Polymeric micelles have been widely explored preclinically as suitable delivery systems for poorly soluble chemotherapeutic drugs in cancer therapy. The present study reported the development of cholesterol (Ch)-conjugated poly(D,L-Lactide) (PLA)-based polymeric micelles (mPEG-PLA-Ch) for effective encapsulation and delivery of curcumin (CUR) at the tumor site. Cholesterol conjugation dramatically affected the particle size and improved drug loading (DL) and encapsulation efficiency (EE). mPEG-PLA-Ch-CUR showed bigger hydrodynamic diameter (104.6 ± 2.1 nm, and 169.3 ± 1.52 nm for mPEG-PLA and mPEG-PLA-Ch, respectively) due to increased size of the hydrophobic core. The newly developed polymer exhibited low critical micelles concentration (CMC) (25 µg/mL) which is close to lipid-based polymer, PEG-phosphatidyl ethanolamine (12.5 µg/mL) compared to mPEG-PLA (50 µg/mL). mPEG-PLA-Ch micelles exhibited relatively higher EE (93.74 ± 1.6%) and DL (11.86 ± 0.8%) compared to mPEG-PLA micelles (EE 91.89 ± 1.2% and DL 11.06 ± 0.8%). mPEG-PLA-Ch micelles were internalized by the cancer cells effectively and exhibited higher cytotoxicity compared to free CUR in both, murine melanoma (B16F10) and human breast cancer (MDA-MB-231) cells. mPEG-PLA-Ch exhibited satisfactory hemocompatibility indicating their potential for systemic application. Further, mPEG-PLA-Ch-CUR demonstrated higher rate of reduction of tumor volume in B16F10-xenografted tumor-bearing mice compared to free CUR. At the end of 22 days, the tumor reduced to 1.87-fold (627.72 ± 0.9 mm3 versus 1174.68 ± 1.64 mm3) compared to the treatment with free CUR. In conclusion, the experimental data in vitro and in vivo indicated that the newly developed CUR-mPEG-PLA-Ch micelles may have promising applications in solid tumors.

Emu oil based nano-emulgel for topical delivery of curcumin.

Curcumin and emu oil derived from emu bird (Dromaius novaehollandiae) has shown promising results against inflammation. However, the delivery of curcumin is hindered due to low solubility and poor permeation. In addition, till date the role of emu oil in drug delivery has not been explored systemically. Hence, the current investigation was designed to evaluate the anti-inflammatory potential of curcumin in combination with emu oil from a nanoemulgel formulation in experimental inflammation and arthritic in vivo models. Nanoemulsion was prepared using emu oil, Cremophor RH 40 and Labrafil M2125CS as oil phase, surfactant and co-surfactant. The optimized curcumin loaded nanoemulsion with emu oil was incorporated into carbopol gel for convenient application by topical route. The anti-inflammatory efficacy was evaluated in carrageenan induced paw edema and FCA induced arthritic rat model in terms of paw swelling, weight indices of the liver and spleen, pathological changes in nuclear factor kappa B, iNOS, COX-2 expression and inflammatory cytokines. Arthritic scoring, paw volume, biochemical, molecular, radiological and histological examinations indicated significant improvement in anti-inflammatory activity with formulations containing curcumin in combination with emu oil compared to pure curcumin. These encouraging results demonstrate the potential of formulations containing curcumin and emu oil combination in rheumatoid arthritis.

An overview of synthetic strategies and current applications of gold nanorods in cancer treatment.

Photothermal therapy, also referred to as optical hyperthermia or photothermal ablation, is an emerging strategy for treating solid tumours. Colloidal gold converts the absorbed light into localized heat via a non-radiative mechanism, surface plasmon resonance, which ablates the solid tumours. Several plasmon resonating nanostructures, including gold nanoparticles (AuNPs), gold nanorods (AuNRs), gold nanoshells, gold nanocages, copper sulphide and carbon nanotubes, have shown potential for photo-activated cancer therapy. Generally, spherical AuNPs display absorption maxima between 500-550 nm, making them inefficient due to low tissue penetration. On the other hand, AuNRs absorb light in the near-infrared (NIR) region that penetrates deeper with higher spatial precision, and causes no damage to the surrounding healthy tissues due to the low energy absorption of NIR light by normal tissue. Moreover, the absorption range of light can be fine-tuned to the NIR region by adjusting the aspect ratios of AuNRs. However, large-scale synthesis and stability of this colloidal system still poses challenges for clinical translation. In this review, we discuss various strategies applied up to now for the synthesis of AuNRs. Current trends in the pre-clinical development of multifunctional AuNRs with emphasis on preparation and application strategies in cancer therapy have been delineated.