Arindam Sen

Transdermal insulin delivery using lipid enhanced electroporation

Transdermal insulin transport by electroporation was measured using porcine epidermis and fluorescein-labeled insulin. Previous studies have shown that anionic lipids can enhance the electroporative transport of molecules up to 10 kDa in size. It was also shown that it is the charge and not the type of the phospholipid head group that influences transdermal transport under electroporation. Moreover, phospholipids with saturated acyl chains enhance the transport of larger molecules more as compared to those with unsaturated chains. In the current study, based on those earlier findings, the effect of 1,2-dimyristoyl-3-phosphatidylserine (DMPS) on the transdermal transport of insulin by electroporation was examined. Porcine epidermis was used as a model for skin. Transport was measured using glass vertical diffusion apparatus in which the epidermis separated the donor and receiver compartments. Negative pulses were applied across the epidermis using platinum electrodes. Results show that when electroporation was carried out in the presence of DMPS, there was greater than 20-fold enhancement of insulin transport. Furthermore, while in the presence of the phospholipid, almost all the transported insulin was detected in the receiver compartment; in the absence of added lipids, only about half the insulin transported was in the receiver compartment and an almost equal amount of insulin remained in the epidermis. Fluorescence microscopy revealed that the insulin transport was mainly through the lipid multilayer regions that surround the corneocytes.

Mild Elevation of Body Temperature Reduces Tumor Interstitial Fluid Pressure and Hypoxia and Enhances Efficacy of Radiotherapy in Murine Tumor Models

Human and rodent solid tumors often exhibit elevated interstitial fluid pressure (IFP). This condition is recognized as a prognostic indicator for reduced responses to therapy and decreased disease-free survival rate. In the present study, we tested whether induction of a thermoregulatory-mediated increase in tissue blood flow, induced by exposure of mice to mild environmental heat stress, could influence IFP and other vascular parameters within tumors. Using several murine tumor models, we found that heating results in a sustained reduction in tumor IFP correlating with increased tumor vascular perfusion (measured by fluorescent imaging of perfused vessels, laser Doppler flowmetry, and MRI) as well as a sustained reduction in tumor hypoxia. Furthermore, when radiation therapy was administered 24 hours postheating, we observed a significant improvement in efficacy that may be a result of the sustained reduction in tumor hypoxia. These data suggest, for the first time, that environmental manipulation of normal vasomotor function is capable of achieving therapeutically beneficial changes in IFP and microvascular function in the tumor microenvironment.

Utilizing temperature-sensitive association of Pluronic F-127 with lipid bilayers to control liposome^cell adhesion

The temperature sensitive properties of Pluronic F-127 (MW approximately 12600, PEO(98)-PPO(67)-PEO(98)), a block co-polymer or poloxamer, was used to control liposome-cell adhesion. When associated with liposomes, the PEO moiety of the block co-polymer is expected to inhibit liposome-cell adhesion. Liposomes were made using egg phosphatidylcholine and different mole% of Pluronic F-127. Size measurement of the liposomes at different temperatures, in the presence and absence of Pluronic F-127, shows significant reduction in the size of multilamellar vesicles, at higher temperatures, by the Pluronic molecules. Negative stain electron microscopy study showed the presence of individual molecules and micelles of Pluronic, respectively at temperatures below and above the critical micellar temperature (CMT). Measurement of the surface associated Pluronics indicated that they associated with liposomes when the sample was heated above the Pluronic CMT, and dissociated from liposomes when cooled below the CMT. Attachment of the Pluronic containing liposomes to CHO cells was inhibited at temperatures above the CMT, but not at temperatures below CMT, indicating that temperature-sensitive control of liposome-cell adhesion is achieved.

Temperature-controlled content release from liposomes encapsulating Pluronic F127.

Temperature-dependent internal content release from liposomes was examined using di-oleoylphosphatidylcholine (DOPC)/cholesterol liposomes with encapsulated Pluronic F127 molecules. The interaction of Pluronic F127 with the lipid bilayer at elevated temperature causes the release of encapsulated contents. Content release was measured using fluorescent markers of two different sizes: small, carboxyfluorescein (CF), and large, bovine serum albumin-conjugated fluorescein iso-thiocyanate (BSA-FITC). Release of CF was studied using fluorescence de-quenching, while that of BSA-FITC was studied using fluorescence emission quenching due to fluorescence resonance energy transfer (FRET). Temperature-controlled complete internal content release was achieved at a precise temperature by controlling the concentration of the encapsulated Pluronic. Increasing cholesterol % in the liposome composition resulted in a sharper transition with temperature in content release. The onset temperature of content release increased with decrease in Pluronic concentration. For the same Pluronic concentration, the onset temperature also depended on the size of the encapsulated marker and was higher for larger markers. We have established that onset of content release is determined by the critical micellar temperature (CMT) of the Pluronic. Temperature-sensitive liposomes, made stealth using di-stearoyl(polyethylene glycol 5000) phosphatidylethanolamine (DSPEG5000PE) in conjunction with Pluronic F127, had similar temperature sensitivity and efficiency in content release compared to the non-stealth liposomes.

The effect of calcium on the bilayer stability of lipids from bovine rod outer segment disk membranes

The phase behavior of bovine rod outer segment disk lipids has been investigated using freeze-fracture and 31P nuclear magnetic resonance (NMR) techniques. 31P-NMR spectra of isolated disk membranes were taken as a function of temperature between 25 degrees C and 45 degrees C. The 31P-NMR spectrum characteristic of phospholipid bilayers was observed at all temperatures both in the absence of Ca2+ and in the presence of 10 mM and 50 mM Ca2+. A similar study was performed on lipids isolated from the disk membranes. In the absence of Ca2+ only lamellar phase behavior was observed. In the presence of less than 10 mM Ca2+, however, there was a change in morphology to non-lamellar structures. Removal of the Ca2+ caused the system to reassume the lamellar form.

Enhanced transdermal transport by electroporation using anionic lipids

Transdermal drug delivery is an attractive approach for either local or systemic treatment in medicine. In the last decade, different active transdermal delivery methods have been further investigated such as cationic liposomal delivery and electroporation-enhanced delivery. In light of gaining a synergistic effect of lipid and electroporation, a new method of using anionic lipids to enhance the transdermal transport of molecules under electroporation is reported here. Heat-stripped porcine epidermis was used for measurement of transdermal transport using an in vitro vertical diffusion apparatus. Lipid vesicles were prepared using a 1:1 mole ratio mixture of 1,2-dioleoyl-3-phosphatidylglycerol (DOPG) and 1,2-dioleoyl-3-phosphatidylcholine (DOPC). When the lipids were mixed with (but not encapsulating) the transport target molecule, the electroporation-induced transport through porcine epidermis was increased as compared to that without the lipids. The enhancement in transport was dependent upon the size and the charge of the transported molecule. Methylene blue (MB), protoporphyrin IX (PpIX) and dimethyl-protoporphyrin IX (DM-PpIX) were used as small target molecules, and FITC-dextrans (4 to 155 kDa) were used as large target molecules in our studies. Enhancement of transport, to varying degree, was observed for all three small molecules (molecular weights <1 kDa), in the presence of DOPG:DOPC vesicles. In the case of large molecules, lipid-enhanced transport was only observed for the 4 kDa dextran, and not for the larger ones (M(w)>10 kDa). Neutral or cationic lipids alone did not enhance the transdermal transport under the electroporation conditions we used.

Saturated Anionic Phospholipids Enhance Transdermal Transport by Electroporation

Anionic phospholipids, but not cationic or neutral phospholipids, were found to enhance the transdermal transport of molecules by electroporation. When added as liposomes to the milieus of water-soluble molecules to be delivered through the epidermis of porcine skin by electroporation, these phospholipids enhance, by one to two orders of magnitude, the transdermal flux. Encapsulation of molecules in liposomes is not necessary. Dimyristoylphosphatidylserine (DMPS), phosphatidylserine from bovine brain (brain-PS), dioleoylphosphatidylserine (DOPS), and dioleoylphosphatidylglycerol (DOPG) were used to test factors affecting the potency of anionic lipid transport enhancers. DMPS with saturated acyl chains was found to be a much more potent transport enhancer than those with unsaturated acyl chains (DOPS and DOPG). There was no headgroup preference. Saturated DMPS was also more effective in delaying resistance recovery after pulsing, and with a greater affinity in the epidermis after pulsing. Using fluorescent carboxyl fluorescein and fluorescein isothiocyanate (FITC)-labeled Dextrans as test water-soluble molecules for transport, and rhodamine-labeled phospholipids to track anionic phospholipids, we found, by conventional and confocal fluorescence microscopy, that transport of water-soluble molecules was localized in local transport spots or regions (LTRs) created by the electroporation pulses. Anionic phospholipids, especially DMPS, were located at the center of the LTRs and spanned the entire thickness of the stratum corneum (SC). The degree of saturation of anionic phospholipids made no difference in the densities of LTRs created. We deduce that, after being driven into the epidermis by negative electric pulses, saturated anionic phospholipids mix and are retained better by the SC lipids. Anionic lipids prefer loose layers or vesicular rather than multilamellar forms, thereby prolonging the structural recovery of SC lipids to the native multilamellar form. In the presence of 1 mg/ml DMPS in the transport milieu, the flux of FITC-Dextran-4k was enhanced by 80-fold and reached 175 microg/cm(2)/min. Thus, the use of proper lipid enhancers greatly extends the upper size limit of transportable chemicals. Understanding the mechanism of lipid enhancers enables one to rationally design better enhancers for transdermal drug and vaccine delivery by electroporation.

Localized delivery to CT-26 tumors in mice using thermosensitive liposomes

A heat-sensitive liposomal drug delivery system was tested using Colon-26 (CT-26) cultured cells and tumors in mice. Lucifer yellow iodoacetamide (LY) was used as a fluorescence marker. The heat-sensitive liposomes exploit the temperature-dependence of critical micellar concentrations of the poloxamer, F127. LY release from unilamellar liposomes at different temperatures was measured. Onset of LY release occurred near 33 degrees C, and reached plateau above 42 degrees C when 90% of the LY was released. Temperature-treated liposomes were mixed with CT-26 cells to measure the binding of the released LY to cell surface. Temperature-dependency of cell-bound LY corresponds to the release curve. CT-26 tumors were grown subcutaneously in both hind legs of Balb/c mice. Mice received heat-sensitive or plain liposomes via tail vein injections, or no liposomes. For each mouse, one tumor was kept at 31.5 degrees C, while the counterlateral tumor was heated to 42 degrees C during injection and for 30min after. LY released in tumors was determined from fluorescence intensity. Tumors receiving heat-sensitive liposomes plus heat treatment showed 2.5-fold greater fluorescence than all other tumors, which were at the background level. This study demonstrates the possible use of poloxamer-containing liposomes as a heat-sensitive drug delivery system in vivo.

Temperature-Dependent Electrical and Ultrastructural Characterizations of Porcine Skin upon Electroporation

The mechanism of high-voltage pulse-induced permeabilization of the stratum corneum, the outer layer of the skin, is still not completely understood. It has been suggested that joule heating resulting from the applied pulse may play a major role in disrupting the stratum corneum. In this study, electrical and ultrastructural measurements were conducted to examine the temperature dependence of the pulse-induced permeabilization of the stratum corneum. The stratum corneum resistance was measured using a vertical diffusion holder, with the stratum corneum placed between two electrode-containing chambers. The stratum corneum resistance was reduced manyfold during the applied pulse. The extent of resistance reduction increased with pulse voltage until reaching a threshold value, above which the resistance reduction was less dependent on the pulse voltage. The stratum corneum was more susceptible to permeabilization at high temperature, the threshold voltage being lower. The stratum corneum resistance recovered within milliseconds after a single 0.3-ms pulse. High-temperature samples had a more prolonged recovery time. Using time-resolved freeze fracture electron microscopy, aggregates of lipid vesicles were observed in all samples pulsed above the threshold voltage. The sizes and fractional areas occupied by aggregates of lipid vesicles at 4 degrees C and at 25 degrees C were measured at different time points after the applied pulse. Aggregates of vesicles persisted long after the electric resistance was recovered. After pulsing at the same voltage of 80 V, samples at 4 degrees C were found to have slightly more extensive aggregate formation initially, but recovered more rapidly than those at 25 degrees C. The more rapid recovery of the 4 degrees C samples was likely due to a lower supra-threshold voltage. Viscoelastic instability propagation created by the pulse may also play a role in the recovery of the aggregates.

Fever-range whole body hyperthermia increases the number of perfused tumor blood vessels and therapeutic efficacy of liposomally encapsulated doxorubicin.

Purpose: Two major questions were addressed: (1) Can fever-range whole body hyperthermia (FR-WBH) affect the number of perfused tumor blood vessels? (2) Can pre-treatment with FR-WBH improve accumulation or anti-tumor efficacy of doxorubicin or DOXIL (liposomal doxorubicin)? Materials and methods: Perfused blood vessels were visualized by intravenous injection of the fluorescent dye (DiOC7(3)) and the number of labeled vessels in tumors and normal organs of unheated mice and those previously heated to 39.5°C for 6 hours were compared. Using three animal tumor models (one syngeneic murine model and two human tumor xenografts in SCID mice) we also compared tumor growth and amount of intratumoral doxorubicin (given as free drug or as DOXIL) in control mice or those given pre-treatment with FR-WBH. Results: FR-WBH had no effect on the number of CD-31 labeled blood vessels. However, in tumors, but not in normal organs of the same animals, FR-WBH resulted in a significant increase in those blood vessels which could take up dye over a prolonged period of time after heating. There was also an increase in DOXIL uptake in the tumors of mice given FR-WBH prior to drug injection as well as enhanced therapeutic efficacy in all three tumor models. Conclusions: FR-WBH increases the number of perfused blood vessels in tumors over a prolonged period following FR-WBH and thus may be useful for improving tumor targeting of cancer therapeutics. We discuss these data in relation to long-conserved thermoregulatory features in normal vasculature, which may be deficient in tumor vasculature.