Self-Assembling Topical Nanomicellar Formulation to Improve Curcumin Absorption Across Ocular Tissues.

The pathophysiological mechanisms for dry and wet age-related macular degeneration (AMD) involve oxidative stress and increased VEGF release and expression. An ideal drug candidate for both types of AMD is the one which offers significant protection to the retinal cells from oxidative stress and inhibit VEGF release. Curcumin is one such natural product which provides numerous beneficial effects including antioxidant, anti-inflammatory, and anti-VEGF activities and has the potential for the treatment of both types of AMD. The bioavailability of curcumin is negligible due to its poor aqueous solubility. The purpose of this work is to develop an aqueous nanomicellar drop formulation of curcumin (CUR-NMF) for back of the eye delivery utilizing hydrogenated castor oil (HCO-40) and octoxynol-40 (OC-40) to treat AMD. A full factorial design was performed with JMP software analysis to optimize the formulation size, polydispersity index (PDI), entrapment efficiency, loading, and precipitation. MTT and LDH assays on human retinal pigmented epithelial (D407) cells revealed that 5-10 µM CUR-NMF dose is safe for ophthalmic use. Furthermore, CUR-NMF exhibited significant protection of retinal (D407) cells against H2O2-induced oxidative stress. In vitro drug release kinetics suggested a sustained drug release profile indicating a long-term protection ability of CUR-NMF against oxidative stress to retinal cells. In addition, an ELISA suggested that CUR-NMF significantly reduces vascular endothelial growth factor (VEGF) release in D407 cell line, hence diminishes the risk of angiogenesis. Collectively, these results suggest that the proposed CUR-NMF can be tremendously effective in treating both types of AMD.

Multi-Layered Nanomicelles as Self-Assembled Nanocarrier Systems for Ocular Peptide Delivery.

Despite the great potential of peptides as therapeutics, there is an unmet challenge in sustaining delivery of sufficient amounts in their native forms. This manuscript describes a novel nanocarrier capable of delivering functional small peptides in its native form. Self-assembling multi-layered nanomicelles composed of two polymers, polyoxyethylene hydrogenated castor oil 40 (HCO-40) and octoxynol 40 (OC-40), were designed to combine hydrophilic interaction and solvent-induced encapsulation of peptides and proteins. The polymers are employed to encapsulate peptide or protein in the core of the organo-nanomicelles which are further encapsulated with another layer of the same polymers to form an aqueous stable nanomicellar solution. The size of the multi-layered nanomicelles ranges from ~ 16 to 20 nm with zeta potential close to neutral (~ - 2.44 to 0.39 mV). In vitro release studies revealed that octreotide-loaded multi-layered nanomicelles released octreotide at much slower rate in simulated tear fluid (STF) (~ 27 days) compared to PBST (~ 11 days) in its native form. MTT assay demonstrated negligible toxicity of the multi-layered nanomicelles at lower concentrations in human retinal pigment epithelial (HRPE, D407), human conjunctival epithelial (CCL 20.2), and rhesus choroid-retinal endothelial (RF/6A) cells. This work demonstrates an efficient small peptide delivery platform with significant advantages over existing approaches, as it does not require modification of the peptide, is biodegradable, and has a small size and high loading capacity.

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.

Clear, Aqueous Topical Drop of Triamcinolone Acetonide.

The objective of this study was to develop a clear aqueous mixed nanomicellar formulation (NMF) of triamcinolone acetonide (TA) with a combination of nonionic surfactant hydrogenated castor oil 60 (HCO-60) and octoxynol-40 (Oc-40). In order to delineate the effects of drug-polymer interactions on entrapment efficiency (EE), loading efficiency (LE), and critical micellar concentration (CMC), a design of experiment (DOE) was performed to optimize the formulation. In this study, full-factorial design has been used with HCO-60 and OC-40 as independent variables. All formulations were prepared following solvent evaporation and film rehydration method, characterized with size, polydispersity, shape, morphology, EE, LE, and CMC. A specific blend of HCO-60 and Oc-40 at a particular wt% ratio (5:1.5) produced highest drug EE, LE, and smallest CMC (0.0216 wt%). Solubility of TA in NMF improved 20 times relative to normal aqueous solubility. Qualitative 1H NMR studies confirmed the absence of free drug in the outer aqueous NMF medium. Moreover, TA-loaded NMF appeared to be highly stable and well tolerated on human corneal epithelial cells (HCEC) and human retinal pigment epithelial cells (D407 cells). Overall, these studies suggest that TA in NMF is safe and suitable for human topical ocular drop application.

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.

How are we improving the delivery to back of the eye? Advances and challenges of novel therapeutic approaches.

INTRODUCTION: Drug delivery to the back of the eye requires strategic approaches that guarantee the long-term therapeutic effect with patient compliance. Current treatments for posterior eye diseases suffer from significant challenges including frequent intraocular injections of anti-VEGF agents and related adverse effects in addition to the high cost of the therapy. Areas covered: Treatment challenges and promising drug delivery approaches for posterior segment eye diseases, such as age-related macular degeneration (AMD) are summarized. Advances in the development of several nanotechnology-based systems, including stimuli-responsive approaches to enhance drug bioavailability and overcome existing barriers for effective ocular delivery are discussed. Stem cell transplantation and encapsulated cell technology (ECT) approaches to treat posterior eye diseases are elaborated. Expert opinion: There are several drug delivery systems demonstrating promising results. However, a better understanding of ocular barriers, disease pathophysiology, and drug clearance mechanisms is required for better therapeutic outcomes. The stem cell transplantation strategy and ECT approach provide positive results in AMD therapy, but there are a number of challenges that must be overcome for long-term efficiency. Ultimately, there are numerous multidimensional challenges to cure vision problems and a collaborative approach among scientists is required.

Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS.

This study aimed to develop and evaluate an ex vivo eye model for intravitreal drug sampling and tissue distribution of connexin43 mimetic peptide (Cx43MP) following intravitreal injection using the microdialysis technique and LC-MS/MS. An LC-MS/MS method was developed, validated, and applied for quantification of Cx43MP in ocular tissues. Microdialysis probes were calibrated for in vitro recovery studies. Bovine eyes were fixed in a customized eye holder and after intravitreal injection of Cx43MP, microdialysis probes were implanted in the vitreous body. Vitreous samples were collected at particular time intervals over 24 h. Moreover, 24 and 48 h after intravitreal injection ocular tissues were collected, processed, and analyzed for Cx43MP concentrations using LC-MS/MS. The LC-MS/MS method showed good linearity (r 2 = 0.9991). The mean percent recovery for lower (LQC), medium (MQC), and higher quality control (HQC) (0.244, 3.906, and 125 µg/mL) was found to be 83.83, 84.92, and 94.52, respectively, with accuracy ranges between 96 and 99 % and limits of detection (LOD) and quantification (LOQ) of 0.122 and 0.412 µg/mL. The in vitro recovery of the probes was found to be over 80 %. As per microdialysis sample analysis, the Cx43MP concentration was found to increase slowly in the vitreous body up to 16 h and thereafter declined. After 48 h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humor. This ex vivo model may therefore be a useful tool to investigate intravitreal kinetics and ocular disposition of therapeutic molecules after intravitreal injection.

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.

Nanocarrier fabrication and macromolecule drug delivery: challenges and opportunities.

Macromolecules (proteins/peptides) have the potential for the development of new therapeutics. Due to their specific mechanism of action, macromolecules can be administered at relatively low doses compared with small-molecule drugs. Unfortunately, the therapeutic potential and clinical application of macromolecules is hampered by various obstacles including their large size, short in vivo half-life, phagocytic clearance, poor membrane permeability and structural instability. These challenges have encouraged researchers to develop novel strategies for effective delivery of macromolecules. In this review, various routes of macromolecule administration (invasive/noninvasive) are discussed. The advantages/limitations of novel delivery systems and the potential role of nanotechnology for the delivery of macromolecules are elaborated. In addition, fabrication approaches to make nanoformulations in different shapes and sizes are also summarized.

Uptake and Permeability Studies to Delineate the Role of Efflux Transporters.

Chemotherapy is one of the major therapeutic interventions in oncology. Despite numerous advances and intensive research, a large number of patients acquire multidrug resistance (MDR) and no longer respond to chemotherapy. Efflux transporters play a predominant role in mediating MDR. Cellular accumulation (uptake) and permeability studies serve as invaluable methods to detect drug efflux/transport mechanism. These methods are generally performed on transfected cells (e.g., MDCKII-MDR1, MDCKII-MRP2, and MDCKII-BCRP) or cells expressing high amount of intrinsic efflux transporters (Caco-2) utilizing specific inhibitors as positive controls. This chapter presents a method of performing uptake and permeability studies, including the preparation of various buffers required for the study.

Novel Injectable Pentablock Copolymer Based Thermoresponsive Hydrogels for Sustained Release Vaccines.

The need for multiple vaccinations to enhance the immunogenicity of subunit vaccines may be reduced by delivering the vaccine over an extended period of time. Here, we report two novel injectable pentablock copolymer based thermoresponsive hydrogels made of polyethyleneglycol-polycaprolactone-polylactide-polycaprolactone-polyethyleneglycol (PEG-PCL-PLA-PCL-PEG) with varying ratios of polycaprolactone (PCL) and polylactide (PLA), as single shot sustained release vaccines. Pentablock copolymer hydrogels were loaded with vaccine-encapsulated poly lactic-co-glycolic acid nanoparticles (PLGA-NP) or with the soluble vaccine components. Incorporation of PLGA-NP into the thermoresponsive hydrogels increased the complex viscosity of the gels, lowered the gelation temperature, and minimized the burst release of antigen and adjuvants. The two pentablock hydrogels stimulated both cellular and humoral responses. The addition of PLGA-NP to the hydrogels sustained immune responses for up to 49 days. The polymer with a higher ratio of PCL to PLA formed a more rigid gel, induced stronger immune responses, and stimulated effective anti-tumor responses in a prophylactic melanoma tumor model.

Recent Patents on Nanoparticles and Nanoformulations for Cancer Therapy.

Cancer is a major malignancy which has claimed numerous lives worldwide. Despite huge resources being utilized to develop cancer therapeutics, no effective cure has been found so far. Hence there is a need to look at emerging technologies for a solution. Nanoparticle is one such technology that has become feasible and popular in the past few years. Though, it has not emerged as a drug delivery platform of choice for cancer therapeutics it has shown enormous promise. Different types of materials such as polymer, lipid, magnet, metal based nanoparticles have been developed to enhance the effectiveness of current treatment. This manuscript will review different aspects of nanoparticles and recent research advances and patents for treatment of cancer.

Uptake and bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir by nanoparticulate carriers in corneal epithelial cells.

PURPOSE: The objective of this study is to investigate cellular uptake of prodrug-loaded nanoparticle (NP). Another objective is to study bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and d-Val-l-Val-GCV (DLGCV) in human corneal epithelial cell (HCEC) model. METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA) NP encapsulating prodrugs of GCV were formulated under a double emulsion method. Fluorescein isothiocyanate isomer-PLGA conjugates were synthesized to fabricate biocompatible fluorescent PLGA NP. Intracellular uptake of FITC-labeled NP was visualized by a fluorescent microscope in HCEC cells. RESULTS: Fluorescent PLGA NP and non-fluorescent NP display similar hydrodynamic diameter in the range of 115-145 nm with a narrow particle size distribution and zeta potentials around -13 mV. Both NP types showed identical intracellular accumulation in HCEC cells. Maximum uptake (around 60%) was noted at 3 h for NP. Cellular uptake and intracellular accumulation of prodrugs are significantly different among three stereoisomeric dipeptide prodrugs. The microscopic images show that NPs are avidly internalized by HCEC cells and distributed throughout the cytoplasm instead of being localized on the cell surface. Following cellular uptake, prodrugs released from NP gradually bioreversed into parent drug GCV. LLGCV showed the highest degradation rate, followed by LDGCV and DLGCV. CONCLUSION: LLGCV, LDGCV and DLGCV released from NP exhibited superior uptake and bioreversion in corneal cells.

Novel delivery approaches for cancer therapeutics.

Currently, a majority of cancer treatment strategies are based on the removal of tumor mass mainly by surgery. Chemical and physical treatments such as chemo- and radiotherapies have also made a major contribution in inhibiting rapid growth of malignant cells. Furthermore, these approaches are often combined to enhance therapeutic indices. It is widely known that surgery, chemo- and radiotherapy also inhibit normal cells growth. In addition, these treatment modalities are associated with severe side effects and high toxicity which in turn lead to low quality of life. This review encompasses novel strategies for more effective chemotherapeutic delivery aiming to generate better prognosis. Currently, cancer treatment is a highly dynamic field and significant advances are being made in the development of novel cancer treatment strategies. In contrast to conventional cancer therapeutics, novel approaches such as ligand or receptor based targeting, triggered release, intracellular drug targeting, gene delivery, cancer stem cell therapy, magnetic drug targeting and ultrasound-mediated drug delivery, have added new modalities for cancer treatment. These approaches have led to selective detection of malignant cells leading to their eradication with minimal side effects. Lowering multi-drug resistance and involving influx transportation in targeted drug delivery to cancer cells can also contribute significantly in the therapeutic interventions in cancer.

Recent Patents in Pulmonary Delivery of Macromolecules.

Pulmonary delivery is a non-invasive form of delivery that holds tremendous therapeutic promise for topical and systemic administration of several macromolecules. Oral administration of macromolecules has several limitations such as low bioavailability, degradation of drug before reaching circulation and insufficient absorption across intestinal membrane. Administration of macromolecules such as proteins, peptides and nucleic acids via inhalation offers great potential due to the avoidance of first pass metabolism, higher surface area and rapid clinical response. However, delivery of reproducible, uniform and safe doses of inhaled particles remains a major challenge for clinical translation. Recent advances in the fields of biotechnology and particle engineering led to progress in novel pulmonary drug delivery systems. Moreover, significant developments in carriers and delivery devices prevent denaturation of macromolecules and control their release within the lungs. This article reviews the advances in pulmonary drug delivery systems by focusing on the recent patents in delivery of macromolecules. Furthermore, recent patents in gene delivery to the lungs have also been discussed. List of patents included in this review is comprehensive in terms of pulmonary delivery of therapeutics. It includes inventions related to proteins and peptides, DNA therapeutics, siRNA and other genetic materials with therapeutic applications. The diseases targeted by these therapeutic molecules are varied including but not limited to different forms of cancer, respiratory diseases etc.

Interaction Studies of Resolvin E1 Analog (RX-10045) with Efflux Transporters.

PURPOSE: Screening interactions of a resolvin E1 analog (RX-10045) with efflux transporters (P-glycoprotein [P-gp], multidrug resistance-associated protein [MRP2], and breast cancer-resistant protein [BCRP]). METHODS: Madin-Darby canine kidney cells transfected with P-gp, MRP2, and BCRP genes were selected for this study. [3H]-Digoxin, [3H]-vinblastine and [3H]-abacavir were selected as model substrates for P-gp, MRP2, and BCRP. Uptake and permeability studies across cell monolayer in both apical to basal (AP-BL) and BL-AP of these substrates were conducted in the presence of specific efflux pump inhibitors and RX-10045. Cell viability studies were conducted with increasing concentrations of RX-10045. RESULTS: Uptake studies showed a higher accumulation in the presence of inhibitors (GF120918 and ketoconazole for P-gp; MK571 for MRP2; and ß-estradiol for BCRP) as well as RX-10045. Similarly, dose-dependent inhibition studies demonstrated higher accumulation of various substrates ([3H]-digoxin, [3H]-vinblastine, and [3H]-abacavir) in the presence of RX-10045. IC50 values of dose-dependent inhibition of RX-10045 for P-gp, MRP2, and BCRP were 239±11.2, 291±79.2, and 300±42 µM, respectively. Cell viability assay indicated no apparent toxicity up to 350 µM concentration. Enhanced permeability for model substrates was observed in the presence of RX-10045. Uptake studies in human corneal epithelial cells suggest that RX-10045 is a strong inhibitor of organic cation transporter-1 (OCT-1). CONCLUSIONS: In summary, the resolvin analog (RX-10045) was identified as a substrate/inhibitor for efflux transporters (MRP2 and BCRP). Also, RX-10045 appears to be a strong inhibitor/substrate of OCT-1. Novel formulation strategies such as nanoparticles, nanomicelles, and liposomes for circumventing efflux barriers and delivering higher drug concentrations leading to a higher therapeutic efficacy may be employed.

Discovery of novel inhibitors for the treatment of glaucoma.

INTRODUCTION: Glaucoma is a neurodegenerative disease with heterogeneous causes that result in retinal ganglionic cell (RGC) death. The discovery of ocular antihypertensives has shifted glaucoma therapy, largely, from surgery to medical intervention. Indeed, several intraocular pressure (IOP)-lowering drugs, with different mechanisms of action and RGC protective property, have been developed. AREAS COVERED: In this review, the authors discuss the main new class of kinase inhibitors used as glaucoma treatments, which lower IOP by enhancing drainage and/or lowering production of aqueous humor. The authors include novel inhibitors under preclinical evaluation and investigation for their anti-glaucoma treatment. Additionally, the authors look at treatments that are in clinics now and which may be available in the near future. EXPERT OPINION: Treatment of glaucoma remains challenging because the exact cause is yet to be delineated. Neuroprotection to the optic nerve head is undisputable. The novel Rho-associated kinase inhibitors have the capacity to lower IOP and provide optic nerve and RGC protection. In particular, the S-isomer of roscovitine has the capacity to lower IOP and provide neuroprotection. Combinations of selected drugs, which can provide maximal and sustained IOP-lowering effects as well as neuroprotection, are paramount to the prevention of glaucoma progression. In the near future, microRNA intervention may be considered as a potential therapeutic target.

Long-term delivery of protein therapeutics.

INTRODUCTION: Proteins are effective biotherapeutics with applications in diverse ailments. Despite being specific and potent, their full clinical potential has not yet been realized. This can be attributed to short half-lives, complex structures, poor in vivo stability, low permeability, frequent parenteral administrations and poor adherence to treatment in chronic diseases. A sustained release system, providing controlled release of proteins, may overcome many of these limitations. AREAS COVERED: This review focuses on recent development in approaches, especially polymer-based formulations, which can provide therapeutic levels of proteins over extended periods. Advances in particulate, gel-based formulations and novel approaches for extended protein delivery are discussed. Emphasis is placed on dosage form, method of preparation, mechanism of release and stability of biotherapeutics. EXPERT OPINION: Substantial advancements have been made in the field of extended protein delivery via various polymer-based formulations over last decade despite the unique delivery-related challenges posed by protein biologics. A number of injectable sustained-release formulations have reached market. However, therapeutic application of proteins is still hampered by delivery-related issues. A large number of protein molecules are under clinical trials, and hence, there is an urgent need to develop new methods to deliver these highly potent biologics.

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery.

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance ((1)H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by (1)H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.

Molecular expression and functional activity of vitamin C specific transport system (SVCT2) in human breast cancer cells.

The main goal of this study is to investigate the expression of sodium dependent vitamin C transport system (SVCT2). Moreover, this investigation has been carried out to define uptake mechanism and intracellular regulation of ascorbic acid (AA) in human breast cancer cells (MDA-MB231, T47D and ZR-75-1). Uptake of [(14)C] AA was studied in MDA-MB231, T47D and ZR-75-1 cells. Functional parameters of [(14)C] AA uptake were delineated in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was carried out with reverse transcription-polymerase chain reaction (RT-PCR). Uptake of [(14)C] AA was studied and found to be sodium, chloride, temperature, pH and energy dependent in all breast cancer cell lines. [(14)C] AA uptake was found to be saturable, with Km values of 53.85 ± 6.24, 49.69 ± 2.83 and 45.44 ± 3.16 µM and Vmax values of 18.45 ± 0.50, 32.50 ± 0.43 and 33.25 ± 0.53 pmol/min/mg protein, across MDA-MB231, T47D and ZR-75-1, respectively. The process is inhibited by structural analogs (l-AA and d-iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca(++)/calmodulin and protein kinase pathways appeared to play a crucial role in modulating AA uptake. A 626 bp band corresponding to a vitamin C transporter (SVCT2) based on the primer design was detected by RT-PCR analysis in all breast cancer cell lines. This research article describes AA uptake mechanism, kinetics, and regulation by sodium dependent vitamin C transporter (SVCT2) in MDA-MB231, T47D and ZR-75-1 cells. Also, MDA-MB231, T47D and ZR-75-1 cell lines can be utilized as a valuable in vitro model to investigate absorption and permeability of AA-conjugated chemotherapeutics.