Md Raihan Chowdhury, Ph.D.

Background: Hypoxia-driven accumulation of extracellular adenosine suppresses anti- tumor immune responses in the tumor microenvironment. Adenosine signals through cAMP-elevating adenosine receptors A2AR/A2BR, triggering downstream PKA-pCREB- mediated signaling that inhibits multiple effector functions of T cells. Recent clinical studies targeting the hypoxia-adenosinergic signaling axis have demonstrated positive responses against various cancers. In this study, we aimed to establish that… Show more

Background: Hypoxia-driven accumulation of extracellular adenosine suppresses anti- tumor immune responses in the tumor microenvironment. Adenosine signals through cAMP-elevating adenosine receptors A2AR/A2BR, triggering downstream PKA-pCREB- mediated signaling that inhibits multiple effector functions of T cells. Recent clinical studies targeting the hypoxia-adenosinergic signaling axis have demonstrated positive responses against various cancers. In this study, we aimed to establish that BWC2094, a novel dual A2A/A2B receptor antagonist, improves T cell responses and enhances the efficacy of current cancer immunotherapies. The extent of CREB phosphorylation driven by the binding of BWC2094 to peripheral blood cells of patients can be used as a metric to propose an efficacious dose. Show less

Purpose:
To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF).

Conclusion:
Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated… Show more

Purpose:
To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF).

Conclusion:
Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated metastases increasing Th1, CTL and DCs infiltration. Show less

Purpose:
To investigate the effect of sub-ablative hyperthermia on PD-1/PD-L1 axis in various cancer cells/tumor models.

Results:
The PD-L1 supernatant evaluated with ELISA demonstrates maximal sPD-L1 modulation in comparison to control at day 3, 5 and 7-9 at a treatment of 45⁰C x 15 mins, 43⁰C x 30 mins and 41⁰C x 60 mins, respectively. The remainder of the treatment combinations did not result in significant modulation or caused cell death. When all kinds of cells (R3230, Hepa… Show more

Purpose:
To investigate the effect of sub-ablative hyperthermia on PD-1/PD-L1 axis in various cancer cells/tumor models.

Results:
The PD-L1 supernatant evaluated with ELISA demonstrates maximal sPD-L1 modulation in comparison to control at day 3, 5 and 7-9 at a treatment of 45⁰C x 15 mins, 43⁰C x 30 mins and 41⁰C x 60 mins, respectively. The remainder of the treatment combinations did not result in significant modulation or caused cell death. When all kinds of cells (R3230, Hepa 1-6 & Hela) were exposed to identified effective thermal doses, a significant modulation of cMet and PD-L1 expression was observed compared to control. For instance, HeLa cells exposed to variable doses of sub-ablative HT demonstrate statistically significant upregulation of PD-L1/c-MET in HT43 and HT45 treated cells at 7 days post treatment (p<0.03) when compared to control or HT41. Indeed, tumors treated in-vivo with 45°C for 15 mins demonstrate statistically significant increased infiltration of PD-1+ cells, and upregulation of PD-L1 and c-MET when compared to sham groups, p <0.05 for all comparisons.

Conclusion:
The sub-ablative HT could result in PD-L1 and cMet modulation in tumors of variable origins (hepatoma, breast cancer, cervical cancer, CRC) and different species that could be targeted with PD-L1 or/& cMet inhibition to induce a favorable response of immune checkpoint inhibitor in cancer treatment.

Clinical relevance:
The sub-ablative HT could modulate PD-L1 & cMet expression on cancer cells that can be targeted with PD-1/PD-L1 axis inhibition for immune check point inhibitor mediated cancer treatment. Show less

Purpose:
To investigate the effect of variable thermal doses on PD-L1 expression in a breast cancer model.

Results:
RF alone 70 x 5 min significantly decreased PD-L1 expression at 7d in the tumor periablational rim as well as 2 HPF away from the rim (Sham: 54±8.1 vs RFA:32.7±3.9 cells/rim, 32.5±4.9 cells/hpf; p<0.001). Additionally, in-vitro sPD-L1 was markedly decreased at 7d in HT41⁰C (60min) (Control:100 vs 69.33±20.9; p<0.05) and at 4d in HT45⁰C (15min) (Control: 100… Show more

Purpose:
To investigate the effect of variable thermal doses on PD-L1 expression in a breast cancer model.

Results:
RF alone 70 x 5 min significantly decreased PD-L1 expression at 7d in the tumor periablational rim as well as 2 HPF away from the rim (Sham: 54±8.1 vs RFA:32.7±3.9 cells/rim, 32.5±4.9 cells/hpf; p<0.001). Additionally, in-vitro sPD-L1 was markedly decreased at 7d in HT41⁰C (60min) (Control:100 vs 69.33±20.9; p<0.05) and at 4d in HT45⁰C (15min) (Control: 100 vs 72.4±15.5; p<0.05). Furthermore, flow cytometry demonstrated markedly downregulated PD-L1 expression in HT45⁰C (15min) compared to control at the day treatment (45.3±6.5 vs 77.9±17.8, %; p<0.05). The PD-L1 expression was slightly upregulated at 2d (all treated groups vs control, but not significant) and then tended to normalize from 4d to 9d. Furthermore, HT45⁰C (15 min) showed relatively downregulated PD-L1 expression at 7d but significant at 9d compared to control (17.7±3.1 vs 39.0±7.1, %; p<0.05). No significant difference in PD-L1 expression was observed in HT41⁰C (60 min) and HT43⁰C (30 min) in all condition.

Conclusion:
The variable thermal doses at different time duration could have significant effects (upregulating and/downregulating) on intralesional PD-L1 expression on cancer cells that could be a targeting strategy for hyperthermia-based cancer treatment.

Clinical relevance:
Hyperthermia could induce PD-L1 expression on cancer cells that can be targeted for hyperthermia-based cancer treatment in combination with nanodrug platform. Show less

Purpose:
Intratumoral APCs such as dendritic cells (DCs) [1] and tumor-associated macrophages (TAMs) [2] play a key role in initiation of anti-tumor antigen specific immunity especially when polarized to mature CD80 positive (DCs) vs immature phenotypes and pro-inflammatory phenotypes (M1) vs tissue healing phenotypes (M2). Thermal tumor ablation (with RFA) can induce increased and altered intratumoral immune cell trafficking. However, variable thermal doses have been shown to induce… Show more

Purpose:
Intratumoral APCs such as dendritic cells (DCs) [1] and tumor-associated macrophages (TAMs) [2] play a key role in initiation of anti-tumor antigen specific immunity especially when polarized to mature CD80 positive (DCs) vs immature phenotypes and pro-inflammatory phenotypes (M1) vs tissue healing phenotypes (M2). Thermal tumor ablation (with RFA) can induce increased and altered intratumoral immune cell trafficking. However, variable thermal doses have been shown to induce different intratumoral cellular infiltration patterns [3]. Here, we investigate the effect of variable thermal doses on intratumoral trafficking of DCs and TAMs following ablation.

Results:
All RFA increased DCs infiltration when compared to sham (51.9±7.6, 52.1±11.3, 30.7±7.0% vs. 19.0±3.4%; p< 0.03). Degree of infiltration was dose dependent with low- and intermediate RFA significantly greater than high dose RFA (p<0.03). Interestingly, percentage of CD80+ DCs dropped with intermediate and high-dose RFA when compared with sham (11.9±2.2, 7.2±1.8 vs 26.0±3.3; p<0.004) however was maintained with low dose 33.7±39.3 (p>0.05). For TAMs, Low and high-dose RFA decreased M2 infiltration when compared to sham. Interestingly, low-dose RFA was the only RFA dose that resulted in improved M1/M2 ratio when compared to sham (29.8±12.8 vs. 3.2±0.4). No significant differences in the percentage of M1 cells were observed between groups (p > 0.05).

Conclusion:
Low-dose RFA improves APC trafficking patterns by increasing intratumoral DCs infiltration while maintaining CD80 positivity, as well as decreasing M2 polarization and improving M1/M2 ratio. Animal survival studies are required to assess overall impact of improved APC infiltrating patterns on oncologic outcomes. Show less

Purpose:
Intratumoral TILs play a key role in mediating and executing anti-tumor antigen specific immunity especially when polarized to Cytotoxic-T-Lymphocytes (CTLs) vs. T-regulator cells (T-regs). Thermal tumor ablation (with RFA) can induce increased and altered intratumoral immune cell trafficking. However, variable thermal doses have been shown to induce different intratumoral cellular infiltration patterns. Here, we investigate the effect of variable thermal doses on intratumoral… Show more

Purpose:
Intratumoral TILs play a key role in mediating and executing anti-tumor antigen specific immunity especially when polarized to Cytotoxic-T-Lymphocytes (CTLs) vs. T-regulator cells (T-regs). Thermal tumor ablation (with RFA) can induce increased and altered intratumoral immune cell trafficking. However, variable thermal doses have been shown to induce different intratumoral cellular infiltration patterns. Here, we investigate the effect of variable thermal doses on intratumoral trafficking of TILs following ablation.

Results:
High dose RFA increased activated CTLs (CD3, CD8, granzyme B) vs. low, intermediate dose - RFA and sham (3.6±1.3% vs. 1.2±1.1%, 0.3±0.2%, 1.3±0.4%, respectively; p< 0.05, all comparisons). Additionally, all RFA groups increased PD-1 positive CTLs (CD3, CD8, PD-1) vs. sham (55.8±9.6% vs. 30.2±0.7; respectively, p<0.05), with high dose RFA resulting in highest PD-1 positive CTL expression when compared to low and intermediate dose RFA (68±2.2% vs. 51.5±7.6%, 48±3.6%, respectively; p<0.05). No significant differences in the percentage of Tregs were observed between groups (p> 0.05).

Conclusion:
High dose RFA results in upregulation of intratumoral activated CTLs shifting the tumor microenvironment towards an anti-tumor spectrum. It also upregulates PD-1 positive CTLs, a targetable immune check point which can be inhibited in combination with RF to further improve ablation induced local anti-tumor immunity. Show less

Purpose:
We recently demonstrated that combining liposomal GM-CSF (lip-GM-CSF) with thermal ablation increases cell trafficking in the periablational rim and untreated residual tumor. In this study, we investigate the effect of combined thermal ablation/lip-GM-CSF therapy on two sets of cell populations in the TME, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). TILs and TAMs are well established prognosticators of clinical outcomes in cancer patients and… Show more

Purpose:
We recently demonstrated that combining liposomal GM-CSF (lip-GM-CSF) with thermal ablation increases cell trafficking in the periablational rim and untreated residual tumor. In this study, we investigate the effect of combined thermal ablation/lip-GM-CSF therapy on two sets of cell populations in the TME, tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). TILs and TAMs are well established prognosticators of clinical outcomes in cancer patients and promote anti-tumor immunity when polarized towards a pro-inflammatory response (TILs; CD8+ cytotoxic T lymphocytes vs. CD4+ T helper cells, TAMs; M1 inflammatory phenotype vs. M2 tissue healing phenotype).

Results:
Combination RFA/lip-GM-CSF significantly increased infiltration of anti-tumor/pro-immune CD8+ TILs and M1 TAM cells over all other groups, both in the periablational rim (CD8+ at 3d and 7d: 113.3±39.3 and 297.7± 35.5 cells, respectively; M1 at 7d: 123.3± 44.5 cells, P<0.05, all comparisons) and in untreated tumor (CD8+ at 7d: 123.9±55.6 cells; M1 at 3d 321.3±150.1 cells, p<0.05 vs. all group comparisons). To a lesser extent, combination RFA/lip-GM-CSF also increased intratumoral trafficking of pro-tumor CD4+ TILs over RFA alone in the periablational rim (CD4+ at 3d and 7d: 102.5± 25.2 cells and 127.7± 29.2, P<0.05) and of CD4+ and M2 cells in untreated tumor (CD4+ at 7d: 135± 25.7 cells; M2 at 3d: 18.6±3.3 cells, P<0.05). Finally, combination RFA/lip-GM-CSF significantly shifted the cellular composition of the tumor microenvironment towards anti-tumor immunity by decreasing the CD4+/CD8+ ratio in the periablational rim (CD4+/CD8+ at 7d from 2.7±0.8 for RFA vs. 0.7±0.3, P<0.05) and untreated tumor (2.6±0.5 for RFA vs. 1.0±0.5, P<0.05). Combination RFA/lip-GM-CSF maintained an anti-tumoral M1/M2 ratio (comparisons between treatment arms not significant). Show less

Abstract:
Since injection administration for diabetes is invasive, it is important to develop an effective transdermal method for insulin. However, transdermal delivery remains challenging owing to the strong barrier function of the stratum corneum (SC) of the skin. Here, we developed ionic liquid (IL)-in-oil microemulsion formulations (MEFs) for transdermal insulin delivery using choline–fatty acids ([Chl][FAs])—comprising three different FAs (C18:0, C18:1, and C18:2)—as biocompatible… Show more

Abstract:
Since injection administration for diabetes is invasive, it is important to develop an effective transdermal method for insulin. However, transdermal delivery remains challenging owing to the strong barrier function of the stratum corneum (SC) of the skin. Here, we developed ionic liquid (IL)-in-oil microemulsion formulations (MEFs) for transdermal insulin delivery using choline–fatty acids ([Chl][FAs])—comprising three different FAs (C18:0, C18:1, and C18:2)—as biocompatible surface-active ILs (SAILs). The MEFs were successfully developed using [Chl][FAs] as surfactants, sorbitan monolaurate (Span-20) as a cosurfactant, choline propionate IL as an internal polar phase, and isopropyl myristate as a continuous oil phase. Ternary phase behavior, dynamic light scattering, and transmission electron microscopy studies revealed that MEFs were thermodynamically stable with nanoparticle size. The MEFs significantly enhanced the transdermal permeation of insulin via the intercellular route by compromising the tight lamellar structure of SC lipids through a fluidity-enhancing mechanism. In vivo transdermal administration of low insulin doses (50 IU/kg) to diabetic mice showed that MEFs reduced blood glucose levels (BGLs) significantly compared with a commercial surfactant-based formulation by increasing the bioavailability of insulin in the systemic circulation and sustained the insulin level for a much longer period (half-life > 24 h) than subcutaneous injection (half-life 1.32 h). When [Chl][C18:2] SAIL-based MEF was transdermally administered, it reduced the BGL by 56% of its initial value. The MEFs were biocompatible and nontoxic (cell viability > 90%). They remained stable at room temperature for 3 months and their biological activity was retained for 4 months at 4 °C. We believe SAIL-based MEFs will alter current approaches to insulin therapy and may be a potential transdermal nanocarrier for protein and peptide delivery. Show less

Abstract:
Ionic Liquids (ILs) have been a topic of interest in many scientific areas since the mid-1990s and can be classified as potential “green solvents,” especially for use in drug delivery systems (DDSs). Because of the “green” and “designer” properties of ILs, the use of ILs has escalated dramatically in pharmaceutics and medicine. Although virtually an unlimited number of ILs could be produced from diverse sources of organic and inorganic cations and anions, including IL precursors… Show more

Abstract:
Ionic Liquids (ILs) have been a topic of interest in many scientific areas since the mid-1990s and can be classified as potential “green solvents,” especially for use in drug delivery systems (DDSs). Because of the “green” and “designer” properties of ILs, the use of ILs has escalated dramatically in pharmaceutics and medicine. Although virtually an unlimited number of ILs could be produced from diverse sources of organic and inorganic cations and anions, including IL precursors derived from biological sources, special consideration must be given for the implementation of ILs in DDSs. Herein, the focus is on the design of the ILs, rather than their implementation in DDSs, particularly the precursor selection, biocompatibility status for safety (biosafety), and potential for targeted drug delivery. It is possible to design an IL suitable for a DDS with synergistic benefits by achieving the maximum therapeutic efficacy of the drug with minimum undesirable side effects caused by the delivery systems or vehicles. Show less

Abstract:
Lipid-based biocompatible ionic liquids (LBILs) have attracted attention as carriers in transdermal drug delivery systems (TDDSs) because of their lipophilic character. In this study, we report the formulation of a peptide−LBIL complex micro-encapsulated in an oil phase as a potential carrier for the transdermal delivery of leuprolide acetate as a model hydrophilic peptide. The peptide−LBIL complexes were prepared via a water-in-oil emulsion composed of… Show more

Abstract:
Lipid-based biocompatible ionic liquids (LBILs) have attracted attention as carriers in transdermal drug delivery systems (TDDSs) because of their lipophilic character. In this study, we report the formulation of a peptide−LBIL complex micro-encapsulated in an oil phase as a potential carrier for the transdermal delivery of leuprolide acetate as a model hydrophilic peptide. The peptide−LBIL complexes were prepared via a water-in-oil emulsion composed of 1,2-dimyristoyl-sn-glycerol-3-ethyl-phosphatidylcho-
line (EDMPC), a fatty acid (stearic, oleic, and linoleic acid)-based LBIL, and cyclohexane followed by freeze-drying to remove the water and cyclohexane. Then, the peptide−LBIL complexes were
nanodispersed and stabilized in isopropyl myristate (IPM) using sorbitol laurate (Span-20). Ionic-liquid-in-oil nanodispersions (IL/ O-NDs) were prepared with varying weight ratios of LBILs and Span-20 as the surfactant and the cosurfactant, respectively. Show less

Abstract
Human skin contains numerous antigen-presenting cells that are a potential target for several immune-based therapies, including vaccination and cancer immunotherapy. However, the outermost layer of the skin—the stratum corneum—acts as a major physical barrier against the permeation of antigens that have a molecular weight > 500 Da. In this study, an ionic liquid-assisted delivery system (ILDS) was developed, which enabled the successful transdermal delivery of an antigenic… Show more

Abstract
Human skin contains numerous antigen-presenting cells that are a potential target for several immune-based therapies, including vaccination and cancer immunotherapy. However, the outermost layer of the skin—the stratum corneum—acts as a major physical barrier against the permeation of antigens that have a molecular weight > 500 Da. In this study, an ionic liquid-assisted delivery system (ILDS) was developed, which enabled the successful transdermal delivery of an antigenic protein, ovalbumin (OVA), with a toll-like receptor agonist, imiquimod, as an adjuvant, to stimulate a specific immune response. Both the ionic liquids and ILDS were completely biocompatible for topical or transdermal application for therapeutic purposes. The skin permeation of the antigenic protein and adjuvant was found to be significantly enhanced because of the incorporation of a surface-active ionic liquid in the ILDS. An in vivo immunization study showed that there was a high level of OVA-specific IgG antibody production because of the enhanced permeation of the antigen and adjuvant across and into the skin. In a preclusive anticancer study, vaccination through ILDS showed stronger tumor-growth inhibition compared to control group. These results indicated that the ILDS could be a promising strategy for transdermal immunization as future therapeutics. Show less

Abstract:
We report a new series of lipid-based biocompatible ionic liquids (LBILs) consisting of the long-chain phosphonium compound 1,2- dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine as the cation and the long-chain fatty acids stearic acid, oleic acid, or linoleic acid as anions. These materials were found to be completely miscible with many polar and nonpolar organic solvents as well as dispersible in water. These LBILs also exhibited excellent biocompatibility with an artificial… Show more

Abstract:
We report a new series of lipid-based biocompatible ionic liquids (LBILs) consisting of the long-chain phosphonium compound 1,2- dimyristoyl-sn-glycero-3-ethyl-phosphatidylcholine as the cation and the long-chain fatty acids stearic acid, oleic acid, or linoleic acid as anions. These materials were found to be completely miscible with many polar and nonpolar organic solvents as well as dispersible in water. These LBILs also exhibited excellent biocompatibility with an artificial three-dimensional human epidermis model. Show less

Abstract:
uids (ILs) have been used as solvents or materials, or both, in many applications, including phar- maceutics and medicine due to their exceptional properties consisting of the combination of “green” pro- perties with tunable physicochemical and biological properties. The use of ILs in the pharmaceutical industry can address many challenges associated with the use of conventional organic solvents or water. ILs have been established as potential solvents to solubilize many insoluble… Show more

Abstract:
uids (ILs) have been used as solvents or materials, or both, in many applications, including phar- maceutics and medicine due to their exceptional properties consisting of the combination of “green” pro- perties with tunable physicochemical and biological properties. The use of ILs in the pharmaceutical industry can address many challenges associated with the use of conventional organic solvents or water. ILs have been established as potential solvents to solubilize many insoluble or sparingly soluble drugs for formulations or delivery. The use of ILs can also address many of the drawbacks of solid-state drugs, including polymorphism and low solubility, stability, and bioavailability. However, many ILs are inherently toxic, which is the main challenge toward developing IL-based drug formulations and drug delivery systems. The use of second- and third-generation ILs comprising more biocompatible cations and anions, compared with the first-generation ILs, has considerably addressed the toxicity issue. A wide range of biocompatible ILs have been designed to improve the pharmacokinetic and pharmacodynamic properties, as well as the biological activity, of drugs. This review describes the advances in the area of green IL-related research and emphasizes the new conceptual development of ILs in pharmaceutics and medicine. Particular attention is given to the mechanistic knowledge in the synthesis of ILs, as well as to the ecotoxicological and biological impact of biocompatible ILs, stimulating the understanding of innova- tive technologies in IL-based drug delivery systems. Show less

Abstract:
Ionic liquid (IL)-based drug delivery systems have attracted considerable interest owing to their intrinsic tunability and ability to transport small or large molecules through the skin. However, the development of “green” ILs remains challenging. Herein, eight potentially “green” fatty acid-based amino acid ILs
(FAAAE-ILs) were synthesized, and their potency in transdermal drug delivery was investigated using ibuprofen and a peptide drug. The synthesized ILs were characterized… Show more

Abstract:
Ionic liquid (IL)-based drug delivery systems have attracted considerable interest owing to their intrinsic tunability and ability to transport small or large molecules through the skin. However, the development of “green” ILs remains challenging. Herein, eight potentially “green” fatty acid-based amino acid ILs
(FAAAE-ILs) were synthesized, and their potency in transdermal drug delivery was investigated using ibuprofen and a peptide drug. The synthesized ILs were characterized to evaluate their
physicochemical, thermal, and biological (cytotoxicity) properties. The in vitro skin permeability of the synthesized FAAAE-ILs was evaluated through pig skin. All of the FAAAE-ILs are liquid at room temperature and freely miscible with pharmaceuticals-permitted solvents/agents (e.g., isopropyl myristate (IPM), Span-20, and DMSO). In vitro cytotoxicity study showed that the cell viability of
all FAAAE-ILs (10% in IPM) was at least 10 times lower than that for a conventional chemical permeation enhancer (CPE), sodium lauryl sulfate. FAAAE-ILs facilitated excellent ibuprofen solubility through multiple hydrogen bonding interactions between the drug and the ILs. An in vitro permeation study showed that the FAAAE-ILs were more effective in enhancing the permeability of drug molecules than the conventional CPE transcutol. The linoleate-based ILs showed a higher degree of permeation than the oleate- based ILs. Among the linoleate-based ILs and ibuprofen formulations (drug in 10% IL in IPM), the L-proline ethyl ester linoleate ([L-ProEt][Lin])-based formulation exhibited best results, followed by β-alanine ethyl ester linoleate, D-proline ethyl ester linoleate, and L-leucine ethyl ester linoleate after 48 h. Show less

Abstract:
The transdermal delivery of sparingly soluble drugs is challenging due to of the need for a drug carrier. In the past few decades, ionic liquid (IL)‐in‐oil microemulsions (IL/O MEs) have been developed as potential carriers. By focusing on biocompatibility, we report on an IL/O ME that is designed to enhance the solubility and transdermal delivery of the sparingly soluble drug, acyclovir. The prepared MEs were composed of a hydrophilic IL (choline formate, choline lactate, or… Show more

Abstract:
The transdermal delivery of sparingly soluble drugs is challenging due to of the need for a drug carrier. In the past few decades, ionic liquid (IL)‐in‐oil microemulsions (IL/O MEs) have been developed as potential carriers. By focusing on biocompatibility, we report on an IL/O ME that is designed to enhance the solubility and transdermal delivery of the sparingly soluble drug, acyclovir. The prepared MEs were composed of a hydrophilic IL (choline formate, choline lactate, or choline propionate) as the non‐aqueous polar phase and a surface‐active IL (choline oleate) as the surfactant in combination with sorbitan laurate in a continuous oil phase. The selected ILs were all biologically active ions. Optimized pseudo ternary phase diagrams indicated the MEs formed thermodynamically stable, spherically shaped, and nano‐sized (<100 nm) droplets. An in vitro drug permeation study, using pig skin, showed the significantly enhanced permeation of acyclovir using the ME. A Fourier transform infrared spectroscopy study showed a reduction of the skin barrier function with the ME. Finally, a skin irritation study showed a high cell survival rate (>90%) with the ME compared with Dulbecco’s phosphate‐buffered saline, indicates the biocompatibility of the ME. Therefore, we conclude that IL/O ME may be a promising nano‐carrier for the transdermal delivery of sparingly soluble drugs. Show less

Abstract:
Transdermal delivery of drugs is more challenging for drugs that are insoluble or sparingly soluble in water and most organic solvents. To overcome this problem, ionic liquid (IL)-mediated ternary systems have been sug- gested as potential drug carriers. Here, we report potent ternary (IL–EtOH–IPM) systems consisting of bio- compatible ILs, ethanol (EtOH), and isopropyl myristate (IPM) that can dissolve a significant amount of the sparingly soluble drug acyclovir (ACV). The ternary… Show more

Abstract:
Transdermal delivery of drugs is more challenging for drugs that are insoluble or sparingly soluble in water and most organic solvents. To overcome this problem, ionic liquid (IL)-mediated ternary systems have been sug- gested as potential drug carriers. Here, we report potent ternary (IL–EtOH–IPM) systems consisting of bio- compatible ILs, ethanol (EtOH), and isopropyl myristate (IPM) that can dissolve a significant amount of the sparingly soluble drug acyclovir (ACV). The ternary systems were optically transparent and thermodynamically stable with a wide range of IL pertinence. An in vitro drug permeation study showed that the ILs in the ternary systems dramatically enhanced ACV permeation into and across the skin. Fourier Transform Infrared spectro- scopy of the stratum corneum (sc) after treatment with ternary systems showed that the skin barrier function was reduced by disturbance of the regularly ordered arrangement of corneocytes and modification of the surface properties of the sc during permeation. Histological analysis, and skin irritation studies using a reconstructed human epidermis model showed the safety profile of the ternary system, and there were no significant changes in the structures of the sc, epidermis, and dermis. Therefore, ternary systems containing biocompatible ILs are promising for transdermal delivery of insoluble or sparingly soluble drugs. Show less

Abstract:
The development of non-toxic ionic liquid-based active pharmaceutical ingredients (IL-APIs) for effective topical drug delivery is still challenging. The properties of IL-APIs can be boosted up by selecting potential biocompatible cations. Here, we introduced N-methyl-2-pyrrolidone (NMP) as a potent biocompatible counter ion to prepare ionic liquefied drugs for topical drug delivery. The cytotoxicity of NMP cation was investigated using mammalian cell lines (HepG2, NIH3T3 and L929… Show more

Abstract:
The development of non-toxic ionic liquid-based active pharmaceutical ingredients (IL-APIs) for effective topical drug delivery is still challenging. The properties of IL-APIs can be boosted up by selecting potential biocompatible cations. Here, we introduced N-methyl-2-pyrrolidone (NMP) as a potent biocompatible counter ion to prepare ionic liquefied drugs for topical drug delivery. The cytotoxicity of NMP cation was investigated using mammalian cell lines (HepG2, NIH3T3 and L929 cells) and compared with conventional IL-forming cations. The synthesized NMP cation has lower toxicity than that of conventional IL-forming cations. The NMP cation showed at least 3.6, 15.2 and 58.9 times lower toxicity than that of conventional imidazolium, ammonium and phosphonium cations, respectively. The synthesized NMP-based ionic liquid (NMP-IL) was characterized using 1H & 13C NMR, FT-IR, DSC and TGA. NMP-IL showed better physico-thermal stability, enhanced skin penetration, and enriched drug ac- cumulation 2.6 times higher than that of IL [Cho][Ibu] in the target tissue. These results suggested that NMP cat- ion based API-IL can be an effective biocompatible formulation for topical drug delivery by accumulating active drugs in the skin. Show less

We report a one-step emulsification and rapid freeze-drying pro- cess to develop a curcumin–ionic liquid (CCM–IL) complex that could be readily dispersed in water with a significantly enhanced solubility of B8 mg mL􏰀1 and half-life (t1/2) of B260 min compared with free CCM (solubility B30 nM and t1/2 B 20 min). This process using an IL consisting of a long chain carbon backbone as a surfactant, may provide an alternative way of enhancing the solubility of poorly water-soluble drugs.

In order to prevent common hypersensitivity reactions to paclitaxel injections (Taxol), we previously reported an ionic liquid-mediated paclitaxel (IL-PTX) formulation with small particle size and narrow size distribution. The preliminary work showed high PTX solubility in the IL, and the formulation demonstrated similar antitumor activity to Taxol, while inducing a smaller hypersensitivity effect in in vitro cell experiments. In this study, the stability of the IL-PTX formulation was monitored… Show more

In order to prevent common hypersensitivity reactions to paclitaxel injections (Taxol), we previously reported an ionic liquid-mediated paclitaxel (IL-PTX) formulation with small particle size and narrow size distribution. The preliminary work showed high PTX solubility in the IL, and the formulation demonstrated similar antitumor activity to Taxol, while inducing a smaller hypersensitivity effect in in vitro cell experiments. In this study, the stability of the IL-PTX formulation was monitored by quantitative HPLC analysis, which showed that IL-PTX was more stable at 4 °C than at room temperature. The in vivo study showed that the IL-PTX formulation could be used in a therapeutic application as a biocompatible component of a drug delivery system. To assess the in-vivo biocompatibility, IL or IL-mediated formulations were administered intravenously by maintaining physiological buffered conditions (neutral pH and isotonic salt concentration). From in vivo pharmacokinetics data, the IL-PTX formulation was found to have a similar systemic circulation time and slower elimination rate compared to cremophor EL mediated paclitaxel (CrEL-PTX). Furthermore, in vivo antitumor and hypersensitivity experiments in C57BL/6 mice revealed that IL-PTX had similar antitumor activity to CrEL-PTX, but a significantly smaller hypersensitivity effect compared with CrEL-PTX. Therefore, the IL-mediated formulation has potential to be an effective and safe drug delivery system for PTX Show less

The technological utility of active pharmaceutical ingredients (APIs) is enormously improved when they are con- verted into ionic liquids (ILs). API-ILs possess better aqueous solubility and thermal stability than that of solid- state salt or crystalline drugs. However, many such API-ILs are not biocompatible or biodegradable. In the current study, we synthesized a series of IL-APIs using methotrexate (MTX), a potential anticancer prodrug, and biocom- patible IL-forming cations (choline and… Show more

The technological utility of active pharmaceutical ingredients (APIs) is enormously improved when they are con- verted into ionic liquids (ILs). API-ILs possess better aqueous solubility and thermal stability than that of solid- state salt or crystalline drugs. However, many such API-ILs are not biocompatible or biodegradable. In the current study, we synthesized a series of IL-APIs using methotrexate (MTX), a potential anticancer prodrug, and biocom- patible IL-forming cations (choline and amino acid esters). The MTX-IL moieties were characterized through 1H NMR, FTIR, p-XRD, DSC and thermogravimetric analysis. The solubility of the MTX-ILs was evaluated in simulated body fluids (phosphate-buffered saline, simulated gastric, and simulated intestinal fluids). An assessment of the in vitro antitumor activity of the MTX-ILs in a mammalian cell line (HeLa cells) was used to evaluate their cyto- toxicity. The MTX-ILs showed aqueous solubility at least 5000 times higher than that of free MTX and two orders of magnitude higher compared with that of a sodium salt of MTX in both water and simulated body fluids. Impor- tantly, a proline ethyl ester MTX prodrug showed similar solubility as the MTX sodium salt but it provided im- proved in vitro antitumor activity. These results clearly suggest that the newly synthesized API-ILs represent promising potential drug formulations. Show less

ABSTRACT:
Paclitaxel (PTX) injection (i.e., Taxol) has been used as an effective chemotherapeutic treatment for various cancers. However, the current Taxol formulation contains Cremophor EL, which causes hypersensitivity reactions during intravenous administration and precipitation by aqueous dilution. This communication reports the preliminary results on the ionic liquid (IL)-based PTX formulations developed to address the aforementioned issues. The formulations were composed of PTX/… Show more

ABSTRACT:
Paclitaxel (PTX) injection (i.e., Taxol) has been used as an effective chemotherapeutic treatment for various cancers. However, the current Taxol formulation contains Cremophor EL, which causes hypersensitivity reactions during intravenous administration and precipitation by aqueous dilution. This communication reports the preliminary results on the ionic liquid (IL)-based PTX formulations developed to address the aforementioned issues. The formulations were composed of PTX/ cholinium amino acid ILs/ethanol/Tween-80/water. A significant enhancement in the solubility of PTX was observed with considerable correlation with the density and viscosity of the ILs, and with the side chain of the amino acids used as anions in the ILs. Moreover, the formulations were stable for up to 3 months. The driving force for the stability of the formulation was hypothesized to be the involvement of different types of interactions between the IL and PTX. In vitro cytotoxicity and antitumor activity of the IL-based formulations were evaluated on HeLa cells. The IL vehicles without PTX were found to be less cytotoxic than Taxol, while both the IL-based PTX formulation and Taxol exhibited similar antitumor activity. Finally, in vitro hypersensitivity reactions were evaluated on THP-1 cells and found to be significantly lower with the IL-based formulation than Taxol. This study demonstrated that specially designed ILs could provide a potentially safer alternative to Cremophor EL as an effective PTX formulation for cancer treatment giving fewer hypersensitivity reactions.
KEYWORDS: paclitaxel, biocompatible ionic liquids, cremophor-free, antitumor activity, hypersensitivity Show less

The technological utility of active pharmaceutical ingredients (APIs) is greatly enhanced when they are transformed into ionic liquids (ILs). API-ILs have better solubility, thermal stability, and the efficacy in topical delivery than solid or crystalline drugs. However, toxicological issue of API-ILs is the main challenge for their application in drug delivery. To address this issue, 11 amino acid esters (AAEs) were synthesized and investigated as biocompatible counter cations for the poorly… Show more

The technological utility of active pharmaceutical ingredients (APIs) is greatly enhanced when they are transformed into ionic liquids (ILs). API-ILs have better solubility, thermal stability, and the efficacy in topical delivery than solid or crystalline drugs. However, toxicological issue of API-ILs is the main challenge for their application in drug delivery. To address this issue, 11 amino acid esters (AAEs) were synthesized and investigated as biocompatible counter cations for the poorly water-soluble drug salicylic acid (Sal) to form Sal-ILs. The AAEs were characterized using 1H and 13C NMR, FTIR, elemental, and thermogravimetric analyses. The cytotoxicities of the AAE cations, Sal-ILs, and free Sal were investigated using mammalian cell lines (L929 and HeLa). The toxicities of the AAE cations greatly increased with inclusion of long alkyl chains, sulfur, and aromatic rings in the side groups of the cations. Ethyl esters of alanine, aspartic acid, and proline were selected as a low cytotoxic AAE. The cytotoxicities of the Sal-ILs drastically increased compared with the AAEs on incorporation of Sal into the cations, and were comparable to that of free Sal. Interestingly, the water miscibilities of the Sal-ILs were higher than that of free Sal, and the Sal-ILs were miscible with water at any ratio. A skin permeation study showed that the Sal-ILs penetrated through skin faster than the Sal sodium salt. These results suggest that AAEs could be used in biomedical applications to eliminate the use of traditional toxic solvents for transdermal delivery of poorly water-soluble drugs.
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Background
Sputum smear microscopy is fast and inexpensive technique for detecting tuberculosis (TB) in high incidence areas but has low sensitivity. Physical and chemical sputum processing along with centrifugation have been found to show promise in overcoming this limitation. Our objective was to compare the sensitivity of smear microscopy obtained with smears made directly from respiratory specimens to those from concentrated specimens.

Probiotic strains can be isolated from many sources, for human applications one of the main criteria is being mammal origin. Cow milk is an important nutrient source for human. Lot of studies showed that this fluid has beneficial effects on the health of human. One reason of being beneficial is explaining by the microflora of cow milk including beneficial lactic acid bacteria. In this research work, 12 isolate were identified as lactic acid bacteria by biochemical characterization and further… Show more

Probiotic strains can be isolated from many sources, for human applications one of the main criteria is being mammal origin. Cow milk is an important nutrient source for human. Lot of studies showed that this fluid has beneficial effects on the health of human. One reason of being beneficial is explaining by the microflora of cow milk including beneficial lactic acid bacteria. In this research work, 12 isolate were identified as lactic acid bacteria by biochemical characterization and further studied for probiotic activity. Among them 9 lactic acid bacteria was posseses probiotic activity. Six Lactobacilli stains were identified as Lactobacillus gasseri(1),Lactobacillus rhamnosus(1),Lactobacillus fermentum(2),Lactobacillus viridescens(3),Lactobacillus farciminis(1), Lactobacillus buchner(4). All of the bacilli isolates were selected to observe potential probiotic activity. These isolates showed resistance to stomach pH (pH 3.0), tolerance against 0.3% bile salt concentration and antimicrobial activity against Escherichia coli, Salmonella enteritids, Staphylococcus aureus, Listeria monocytogenes. The isolates were used to investigate the tolerances of the isolates to other stress conditions, such as, growth at different NaCl solution, growth at different temperatures and in the presence of 0.5% CaCO3. All of the isolate showed good tolerances in stress conditions. After investigation the probiotic properties of these isolates, they were identified by biochemical characterization techniques. It is observed that, cow milk is a source of potential probiotic strains. Show less

This study describes the chemical investigations of Aegle marmelos (A.marmelos), a plant belonging to the family rutaceae. Although some investigations mainly on leaves and fruits have previously been reported with this plant, but very little investigations on bark of A.marmelos have been reported to date. The stem bark of A. marmelos was extracted with organic solvent and the extracts were fractionated by using standard chromatographic techniques. A total of three compounds were isolated of… Show more

This study describes the chemical investigations of Aegle marmelos (A.marmelos), a plant belonging to the family rutaceae. Although some investigations mainly on leaves and fruits have previously been reported with this plant, but very little investigations on bark of A.marmelos have been reported to date. The stem bark of A. marmelos was extracted with organic solvent and the extracts were fractionated by using standard chromatographic techniques. A total of three compounds were isolated of which two (AM-1 & AM-5) have been isolated from different plant, but this is the first report of their occurrences in the bark of A. marmelos. The isolated pure compounds were identified by extensive analyses of their high resolution Nuclear Magnetic Resonance (1H-NMR) spectral data. The powdered stem bark of A. marmelos was extracted with methanol. Chromatographic (vacuum liquid chromatography) fractionation and purification of the crude methanolic extract yielded compound (AM-1), one coumarin (AM-5), and one sterol (AM-2). The purified compound (AM-1) was identified as Lupeol, compound (AM-5) was identified as 5-Methoxy psoralen and compound (AM-2) was identified as stigmasterol or 24-ethyl-cholesta-5- 22-dien-3beta-ol. Show less

A reliable and sensitive RP-HPLC method has been developed and validated for simultaneous assay of Adapalene & Benzoyl peroxide. An isocratic separation of Adapalene & Benzoyl peroxide was achieved on C18, 250Χ4.6 mm i.d., 5 μm particle size columns with a flow rate of 1.0 ml/min and using a UV detector to monitor the elute at 270 nm. The mobile phase consisted of a mixture of water, acetonitrile, tetrahydrofuran and trifluroacetic acid at a ratio of 29:33:38:0.2 by volume. Application of the… Show more

A reliable and sensitive RP-HPLC method has been developed and validated for simultaneous assay of Adapalene & Benzoyl peroxide. An isocratic separation of Adapalene & Benzoyl peroxide was achieved on C18, 250Χ4.6 mm i.d., 5 μm particle size columns with a flow rate of 1.0 ml/min and using a UV detector to monitor the elute at 270 nm. The mobile phase consisted of a mixture of water, acetonitrile, tetrahydrofuran and trifluroacetic acid at a ratio of 29:33:38:0.2 by volume. Application of the suggested procedures laid on ICH guidlines was successfully applied to the determination of these compounds in active pharmaceutical ingredients and in pharmaceutical preparations, with high percentage of recovery, good accuracy and precision. Response was a linear function of drug concentration in the range of (0.79-1.20) mg/mL range with an R2 of 0.999 for Benzoyl peroxide & (39.68-59.90) μg/mL range with an R2 of 0.999 for adapalene, accuracy with percent RSD of 100.65±0.23 (Benzoic peroxide) & 100.48±0.45 (Adapalene) and with a limit of detection and quantification of 24.74PPM, 92.53 PPM and 0.032 PPM, 0.126 PPM for Benzoyl peroxide & Adapalene respectively. Show less

Diabetes mellitus is a group of metabolic disease characterized by high blood glucose levels that results from defects in insulin secretion or action or both. Some recent studies had shown that elevated level of uric acid is involved in the pathogenesis of type 2 diabetes, regardless of other characteristics of subjects. Our aim was to investigate the association of serum uric acid concentration or level with diabetes mellitus in Bangladeshi population. One hundred and fifty type 2 diabetes… Show more

Diabetes mellitus is a group of metabolic disease characterized by high blood glucose levels that results from defects in insulin secretion or action or both. Some recent studies had shown that elevated level of uric acid is involved in the pathogenesis of type 2 diabetes, regardless of other characteristics of subjects. Our aim was to investigate the association of serum uric acid concentration or level with diabetes mellitus in Bangladeshi population. One hundred and fifty type 2 diabetes mellitus subjects (male 83, female 67) and fifty non-diabetic subjects (25 male, 25 female) were included in the study over six months. Uric acid was measured by enzymatic colorimetric method. The level of uric acid was higher in male and female diabetic subjects (p<0.05 and p<0.001) compared to non-diabetic subjects. Uric acid showed a positive association with fasting blood sugar (FBS), diastolic blood pressure, HbA1c in case of diabetes subjects. These data strongly suggest that compared to non-diabetic subjects, diabetic subjects have significantly higher uric acid level. Show less

Hepatitis B attacks the liver and can cause both acute and chronic disease. Elevated ALT, AST and ALP are the parameter of liver function test. Along with this high alpha fetoprotein serum levels have also been found in 60–70% of patients with hepatocellular carcinoma; nevertheless, there are other causes that increase this particular protein. A prospective study was performed among 32 healthy control (HBsAg-) and 96 patients (HbsAg+) with their first attack of HBV-related acute hepatitis. In… Show more

Hepatitis B attacks the liver and can cause both acute and chronic disease. Elevated ALT, AST and ALP are the parameter of liver function test. Along with this high alpha fetoprotein serum levels have also been found in 60–70% of patients with hepatocellular carcinoma; nevertheless, there are other causes that increase this particular protein. A prospective study was performed among 32 healthy control (HBsAg-) and 96 patients (HbsAg+) with their first attack of HBV-related acute hepatitis. In these study AFP, AST, ALT and ALP levels were measured. Among the 29 patients, 9 of them exhibited AFP level in between 10-20(IU/ml), 8 of them exhibited AFP level in between 21-70(IU/ml) and 12 of them exhibited AFP level in between 71-300(IU/ml) with a highest level of 297.90(IU/ml). Beside those 29 patients, other 67 patients of our 96 patient groups have also showed normal AFP level lower than 10(IU/ml). Show less

Sensitivity and specificity of direct and concentrated AFB smear microscopy has been investigated by PCR analysis and culture methods to determine a rapid and cheap detection of pulmonary tuberculosis. In this study 305 patients were selected and 915 specimens were collected from suspected and hospital admitted patients. Patients were taken from Dhaka Central Jail Hospital and National TB Hospital, Dhaka, Bangladesh. All samples were smeared and Ziehl-Neelsen method and Lowenstein-Jensen (L-J)… Show more

Sensitivity and specificity of direct and concentrated AFB smear microscopy has been investigated by PCR analysis and culture methods to determine a rapid and cheap detection of pulmonary tuberculosis. In this study 305 patients were selected and 915 specimens were collected from suspected and hospital admitted patients. Patients were taken from Dhaka Central Jail Hospital and National TB Hospital, Dhaka, Bangladesh. All samples were smeared and Ziehl-Neelsen method and Lowenstein-Jensen (L-J) method were applied. PCR analysis and culture method was used to confirm the detection. Isolated DNA was used in PCR analysis. In this detection study PCR based IS6110 analysis was developed to identify the pulmonary tuberculosis rapidly and cheaply. A total of 915 samples were prepared for analysis and found 70.47% and 82.85% sensitivity in direct and concentrated smears respectively. Both type of smears (direct and concentrated) showed 100% sensitivity and specificity in culture and PCR IS6110 analysis. Show less

Tuberculosis is a global public health issue and a disease burden in Africa and Asia. Bangladesh is one of the high burden countries in
Southeast Asian region. Mycobacterium tuberculosis the causative agent of tuberculosis, is a devastating bacterium because it spreads
person to person, acquired multiple antibiotic resistance and most importantly has the limitation in rapid diagnosis, specially in case of
extrapulmonary infection. Here we showed Real-Time PCR assay with the primers… Show more

Tuberculosis is a global public health issue and a disease burden in Africa and Asia. Bangladesh is one of the high burden countries in
Southeast Asian region. Mycobacterium tuberculosis the causative agent of tuberculosis, is a devastating bacterium because it spreads
person to person, acquired multiple antibiotic resistance and most importantly has the limitation in rapid diagnosis, specially in case of
extrapulmonary infection. Here we showed Real-Time PCR assay with the primers targeting IS6110 is a better method than conventional
AFB microscopy for the diagnosis of extrapulmonary tuberculosis. For the comparative study 99 extrapulmonary specimens from 9 different
parts of the body were collected from suspected patients. Among those only 10 samples were positive, where 50% them were scanty positive
in AFB microscopy, on the other hand 33 samples were positive in Real–Time PCR assay. All samples positive in AFB microscopy were
also positive in Real-Time PCR assay with an additional 23 positive. Higher percentage of positive results were found in Real-Time PCR
analysis in all the samples except Percutaneous nephrostomy (PCN) fluid and Pus. The sharp differences in the result indicate the
effectiveness and of the Real-Time PCR assay over the conventional AFB microscopy. High precision and accuracy make it better choice as
a diagnostic method for the diagnosis of extrapulmonary tuberculosis. Show less

An examination of adsorption of phenol and phenolic compound on Rice Straw is followed by a critical assessment of low-cost adsorbents. Experiments have been conducted to examine the adsorption of phenol and phenolic compounds from industrial effluent by using agricultural wastes on Rice Straw and were carried out for the analysis of adsorption equilibrium capacities using a batch equilibrium technique. Batch adsorption isotherm studies were carried out under varying experimental conditions of… Show more

An examination of adsorption of phenol and phenolic compound on Rice Straw is followed by a critical assessment of low-cost adsorbents. Experiments have been conducted to examine the adsorption of phenol and phenolic compounds from industrial effluent by using agricultural wastes on Rice Straw and were carried out for the analysis of adsorption equilibrium capacities using a batch equilibrium technique. Batch adsorption isotherm studies were carried out under varying experimental conditions of contact time, operational temperature, adsorbent dose, initial phenol concentration, particle size and pH of phenol solution. Adsorption equilibrium of Rice Straw was reached within 3 hour for phenol concentration 500μg/L. Kinetics of adsorption obeyed a first-order rate equation. The suitability of the Freundlich and Langmuir adsorption models to the equilibrium data were investigated for phenol-sorbent system. The results showed that the equilibrium data for all the phenol-sorbent systems fitted the Freundlich model and Langmuir modal within the concentration range studied. 83.2% removal of phenol were achieved at given adsorption conditions from aqueous system which could be regenerated by desorption with the help of 1 M NaOH and recovered 95.30% of adsorbed phenol.... Show less