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The purpose of this research work was to explore an application of uncoated porous drug carrier prepared by single-step drug adsorption for a delivery system based on integration of floating and pulsatile principles intended for chronotherapy. This objective was achieved by utilizing 32 factorial design, solvent volume (X1) and drug amount (X2) as selected variables, for drug adsorption using solvents, methanol, and dichloromethane (DCM), of varying polarity. Nitrogen adsorption (N2), scanning electron microscopy of cross-sections, and atomic force microscopy were done to study adsorption patterns and their effect on release pattern. Drug release study was customized by performing for 6 h in acidic environment to mimic gastroretention followed by basic environment akin to transit phase. Correlation between porous data from mercury and N2 adsorption was probably studied for the first time. Observed regression analysis values for pore volume, surface area, and drug release indicated the influence of selected variables. Total release range in acidic medium was 12.77–24.57% for methanol, 8.79–15.26% for DCM, and final release of 69.45–92.23% for methanol, and 60.16–99.99% for DCM influenced by varying internal geometries was observed. Present form of drug delivery system devoid of any additives/excipients influencing drug release shows distinct behavior from other approaches/technologies in chronotherapy by (a) observing desired low drug release (8%) in acidic medium, (b) overcoming the limitations of process variables caused by multiple formulation steps and different characteristic polymers, (c) reducing time consumption due to single step process, and (d) extending as controlled/extended release.
During the past decade, there has been an exponential growth in the investigations related to the application of porous material in controlling temporal or distributional drug release by oral, pulmonary, transdermal, and injectables routes. Inherent attractive features, like stable uniform porous structure, high surface area, tunable pore sizes with narrow distribution, and well-defined surface properties, make them suitable for their inclusion in any form of delivery system. Porous network, characteristic of an individual material, is important in determining both natural and practical applications such as adsorption, dissolution, and diffusion of drugs. This allows them to adsorb drugs and release them in a more reproducible and predictable manner. Until today, different types of drugs with small and large molecular size have been evaluated (1–6). Various methods like stirring in drug solution or suspension, immersion for long times until equilibrium is reached, vacuum, emulsion formation, gravimetric method, and solvent evaporation to entrap the drug within the carrier are reported (6–11).
In our previous communications, we had demonstrated the applications of a porous carrier, Accurel MP 1000®, made of isotactic polypropylene, as an uncoated drug carrier (12) and its utility for the development of ‘floating pulsatile drug delivery system’ intended for chronotherapy (13). This therapy, based on circadian rhythm, is contrary to principles of present delivery systems based on providing variable/constant drug amount over a period of time, where timed non-uniform release profile is found more beneficial than a uniform one (14).The essential features of this conceptual delivery system exhibit as a (a) multiparticulate system, (b) combination of gastroretentive and pulsatile principles in same dosage form, (c) single-step formulation process using low-density porous carrier as drug-loading core, (d) idealistic drug release profile that is effective at morning time intended for a particular pathological condition based on chronotherapy, and (e) having low drug release in stomach suited for non-steroidal anti inflammatory drug (NSAID) class of drug (ibuprofen).
The contribution of low-density porous carrier in the development of this drug delivery system associates major significance by (a) ensuring the retention of dosage form in the stomach for an extended period without using any excipients for enhancing floating, (b) simultaneous least release all through this period (resembling lag phase) suited for NSAID drugs to avoid gastric irritation, (c) choice of drug loading (melt and solvent evaporation), and(d) limit/overcome various formulation variables by acting as a drug-loading core using single formulation step when compared with other approaches/methods, which need multiple steps by using various polymers and excipients to achieve such release profile. The implication of last point seem to be realistic not only when compared with our previous reported work using hydrogels for formulating the same delivery system (15) but also with other methods based on different approaches (10,16–20) and technologies like OROS®, CONTIN®, CEFORM®, TIMERx®, etc. (21) developed for chronotherapy.
Successful relevance of formulating this conceptual system reported earlier was done by using just three batches via melt and solvent evaporation method (13). Based on excellent observed results, the need to study/explore further drug adsorption via solvent evaporation for its simplicity along with optimization by employing factorial design was considered, thereby stating as the objective of present work. The same design of experiment was used earlier in another study for ascertaining as a drug carrier, which in turn acted as a source of comparison for understanding various aspects of mass transfer (12). Additional characterization process by drift Fourier transform infrared analysis (FTIR), scanning electron microscopy (SEM) of cross-sectional areas, N2 adsorption, and atomic force microscopy (AFM) was done for better understanding. First attempt to find any correlation between porosimetry data obtained from N2 adsorption by Brunauer–Emmett–Teller (BET) method and mercury porosimetry (12) for drug-loaded porous carrier was done. Drug release study was tailored for 6 h as the maximum gastric floating time to evaluate the concept of floating followed by 3 h in basic medium.
Accurel MP 1000® (Membrana ,Germany), a low-density microporous polypropylene microparticles with particle size <1,500 μm, pore size in the range from 5 to 20 µm, and void volume of 70% and ibuprofen (Cipla, India) were obtained as a generous gift. Methanol (M), dichloromethane (DCM), and other reagents were of analytical grade.
Ibuprofen was loaded onto the porous beads by solvent evaporation. Accurel MP 1000 was closely sieved in the range of 250–350 μm to nullify effect due to variation in particle size. In a typical study, various amounts of drug was dissolved in the multiple volumes of solvent (M or DCM) followed by the constant addition of 100 mg Accurel MP 1000 ®, kept to evaporate solvent under ambient conditions.
A 32 design was employed to find the interaction between the selected variables. The variables chosen were volume of solvent (X1) and the amount of drug (X2) at three different levels. The coded and the actual values of the experimental design are given in Table I. The data analysis of values obtained for pore volume, surface area, pore diameter, and drug release at various intervals from all batches were subjected to multiple regression analysis using statistical software Unistat® (Statistic version 3, Meglon, USA). The equation fitted was
where Y=measured response, X=levels of factors, and β=coefficient computed from the responses of the formulations.
The dried weight of microparticles after drug loading was recorded as final yield. The drug-loaded beads were dissolved in methanol kept on ultrasonicator, and the content of drug content was assayed by determining (in triplicate) the absorption at 221 nm using UV-VIS Spectrophotometer V-500 (Jasco International Co. Ltd., Japan). The experiment was repeated thrice in order to establish accuracy and precision of the method.
Thermograms of ibuprophen and Accurel MP 1000 microparticles with and without drug were obtained using differential scanning calorimeter (Mettler-Toledo DSC 821e, Switzerland) equipped with an intracooler. Indium standard was used to calibrate the temperature and enthalpy scale. The powder samples were hermetically sealed in perforated aluminum pans and heated at constant rate of 10°C/min over the temperature range 25–150°C. The system was purged with nitrogen gas at the rate of 100 mL/min to maintain inert atmosphere.
X-ray powder diffraction (XRD) patterns of drug and Accurel MP 1000® beads with and without drug were recorded by using an X-ray diffractometer (Philips PW 1729, The Netherlands). Samples were irradiated with monochromatized Cu Kα radiation (1.542 Å) and analyzed at 2θ between 2° and 60°. The voltage and current used were 30 kV and 30 mA, respectively. The range and the chart speed were 5×103 CPS and 10 mm/2θ, respectively.
Appropriately weighed drug-loaded microparticles were subjected to gravimetric assay between 35 and 175°C, with the heat flow of 5°C/min using Thermogravimetric Instrument (Seiko TG/DTA-32, Japan). All experiments were performed in the presence of static air.
FTIR measurements of drug, Accurel MP 1000®, and drug-loaded ones were obtained on FTIR spectrophotometer (Spectrum One, Perkin Elmer Instruments, UK). Samples were prepared by mixing with KBr and placing in the sample holder. The spectra were scanned over the wave number range of 3,600 to 400 cm−1 at ambient temperature.
Microphotographs of the cross-sections of beads were observed using scanning electron microscope (Cambridge Stereoscan 120, UK) operated with an acceleration voltage of 10 kV. The beads were mounted on the standard specimen mounting stubs and were coated with a thin layer (20 nm) of gold in sputter coater unit (VG Microtech, UK).
AFM measurements were performed in ambient condition on coated pellets without any further sample preparation. The AFM measurements were performed repeatedly on many different pellets and on several different areas. Although variations were seen, the images presented were typical of those most frequently observed. All the AFM measurements were done in the contact mode using a commercial Multimode with Nanoscope® IV controller (Veeco Instruments Inc., Santa Barbara, CA, USA). Height and deflection images were taken and analyzed using the NanoScope version 5.12r5 software in the offline mode.
Total surface area and the porosity of the Accurel MP 1000® microparticles with and without drug were measured by nitrogen adsorption using a porosimeter (Quantachrome instrument, Autosorb-1TM, gas sorption system, Ontario, Canada). Briefly, weighed amounts of samples were placed in the glass cells and outgassed with nitrogen at 25°C for 3 h before analysis. Subsequently, the sample and the reference cells were immersed in liquid nitrogen at −196°C, and absorption isotherm was obtained from the volume of nitrogen (cm3/g) adsorbed onto the surface as a function to relative pressure. Total surface area was calculated by BET method. Various evaluation parameters were obtained by using Autosorb-1TM software. Mercury intrusion porosimetry was done using Mercury porosimeter (Autoscan 33 USA). Samples were first loaded with mercury in a pressurized cell under vacuum and later subjected to pressure range of 0–33,000 psi (12).
The dissolution of drug-loaded Accurel MP 1000 ® beads was studied using USP XXIV type II dissolution test apparatus (Electrolab TDT-06P, India), containing 900 mL of pH 1.2 HCl and pH 7.2 phosphate buffer maintained at 37±0.5°C and stirred at 100 rpm for 6 and 3 h, respectively. Samples were collected periodically and replaced with a fresh dissolution medium. Data were analyzed using PCP Disso software (v2.08, Poona College of Pharmacy, India). All readings were made in triplicate.
The stability of few selected drug-loaded Accurel MP 1000® batches was monitored up to 3 months at ambient temperature and relative humidity (30°C/60% RH). Samples were removed and characterized by dissolution studies and DSC.
The observed values for yield and drug content are given in Table I. Summary of the earlier reported results entail the influence of solvent nature, volumes, and drug amount on the observed values, which was well supported by statistical analysis. DSC and XRD predicted no change in crystalline nature during adsorption. Influence of pore shielding effect using different solvents suggesting discreet drug adsorption was observed by TGA.
FTIR results are displayed in Fig. 1. Peak values from 2,961 to 2,722 cm−1 of Accurel MP 1000® showing multiple peaks transformed in to sharp triplet peaks after drug adsorption without changing the values of other peaks like 2955, 2922, 2869 cm−1 so on. Same was true for particular drug peaks, which show perfect repetition in the range of 1,507 to 2,173 cm−1. Apart from them, various other peaks also showed excellent reproducibility. Another characteristic peaks around 2,625 and 2,730 cm−1 indicated toward the dimmer formation signifying the interaction of drug over the hydrophobic surface (22). Overall, these results suggest the physical nature of interaction rather than chemical one.
In the pursuit of studying inner/core adsorption behavior for descriptive follow-up, SEM of cross-sections of the microparticles was considered as shown in Fig. 2. Regular spread out adsorption pattern using methanol was observed in Fig. 2b–d. This can be related to lower viscosity and slow phase transformation caused by high boiling point of solvent. At low solvent volume, the drug adsorption is slightly concentrated in upper layers, which change with increasing solvent volume by showing migration toward the core as seen in Fig. 2c and d. This demonstrates the interactive and mobile behavior of drug, having both hydrophilic and hydrophobic segments, over hydrophobic polypropylene surface, whereas polar interactions take place between adsorbed molecules of methanol and those in the solution resulting in observed adsorption orientation. The magnitude of these interactions can show dependence upon free energy of solid and the dispersed adsorbate (22). On the contrary, DCM, having low boiling with low polarity, indicating fast phase change, depicts exactly an opposite pattern than observed with methanol (Fig. 2e–h). These characteristic pictures reveal the concentric adsorption at the surface in the form of stakes running throughout. The partial migration can be due to the decreased polarity of solvent limiting wetting of hydrophobic surface (23). Higher magnification reveal the hemispherical adsorption behavior reported earlier using less polar solvent with hydrophobic surface (24).
AFM was done for further evaluation of the surface morphology for any difference in adsorption pattern as seen in Figs. 3 and and4.4. A contact mode AFM measurement of batch 7 using methanol in air was observed (Fig. 3a–f). All the corresponding images and 3D plot suggest the true character of the surface. The surface shows wavy nature, which is indicated by the light and dark colors in the images, having irregular patterns caused by depressions and counters. Pictures revealed cracks and height conditions of around 1 μm with a non-regular pattern. Cracks were found even at high magnifications, which suggest the discontinuity of the adsorbed drug layer. This type of surface may be due to the underlying texture of the core structure and/or due to various drying phenomenon occurring during solvent evaporation governed by capillary and diffusion (25). Other factor that surfaced can be due to the interactions taking place using high polar solvent with hydrophobic surface as discussed earlier.
With batch 7, using DCM revealed a large concentric area of adsorbed drug with a considerable difference in the layout of adsorbed drug pattern, as seen in Fig. 4a. The large chunk of adsorbed drug area suggests the formation of stakes, thereby showing a large depth on all sides. On further magnification, the wavy nature becomes more prominent. The size of surface taken at 3×3 μm (Fig. 4g–i) and 1×1 μm (Fig. 4j–l) shows the layered form of adsorption phenomenon suggested by a number of counters with a stepwise formation. No cracks in the adsorbed drug pattern were observed, indicating its continuity over the surface. This supports the claim and explanation of interaction between the low polarity solvent DCM with the hydrophobic surface/pores on the adsorption pattern (24).
The drug-loading process influencing surface area and porosity was studied by adsorbing N2 whose values are given (Table II). All isotherms were of type III showing hysteresis displayed due adsorption and desorption in mesopores (24). Using methanol, recorded values for all evaluation parameters showed influence of selected variables. With least solvent volume (1 ml), observed values decreased with increasing drug amount, which differed with increasing solvent volume irrespective of drug amount used. The reason for this can be attributed to the impact of variable solvent volumes, wherein capillary (at low-solvent volume) or pressure gradient (at high-solvent volumes leading to submerging) or both work in manipulating the adsorption pattern (26). This was evident from the graphs, which show increased activity below 50 Å with increasing solvent volume (supplementary data). However, batches using 200 mg drug showed high values using increased solvent volumes. This feature was not seen using mercury porosimeter, which followed a particular trend (12).
Using DCM, recorded values for all evaluation parameters showed varied patterns at different drug levels. Surface area decreased with increasing solvent volume at lowest amount of drug but with minor difference, which behaved oppositely at other two levels (200 and 300 mg). Negligible difference between the values was observed. This indicates the continuous adsorption pattern on the surface at increased drug amount where less/no accessibility of nitrogen gas was possible, thereby reconfirming our previous results. Once again, this sort of result was not obtained using mercury porosimetry.
Statistical interpretation of the data by regression analysis is given in Table III. Using methanol, observed values display a non-significant negative impact of drug–drug interaction (β2) for surface area and positive drug interaction (β) for pore volume along with negative impact of drug–solvent (αβ) interaction. This distantly points toward the possibility of interactions of highly polar nature of solvent with drug containing both hydrophobic and hydrophilic groups on hydrophobic surface of Accurel MP 1000®. Using DCM, drug amounts (β) predict negative influence over surface area (Fig. 5a) and pore volume (Fig. 5b). The closely packed drug at the periphery of the surface may have hindered the nitrogen, thereby showing negative influence, while with methanol, the observed adsorption pattern must have worked conversely. Drug–drug interaction shows positive values for both response variables using DCM and negative response for surface area using methanol. This can be related to modified pore sizes and geometry after drug adsorption, which is more limited to outer surface using DCM and spread throughout using methanol.
Combined N2 adsorption and mercury-intrusion porosimetry experiments on various porous materials have indicated a partial agreement concerning various aspects of evaluation parameters. Drug-loaded porous materials showed distinct difference and similarity of statistical data on comparing the present data with raw mercury porosimetry data conducted in our previous work. Values related to the amount of drug variable (β) showed its influence during MPA only while solvent–solvent interaction was absent for both. The difference between the analysis of mercury porosimetry data (12) and nitrogen sorption data can be correlated to the (a) different methodology basis of both techniques for their evaluation, (b) shape of individual pores after drug loading, (c) type of pore openings, (d) pore blockages, (e) relation between voids, and throat (f) the cooperative percolation effect of the porous network.
Basic theoretical background of drug release from hydrophobic porous polymeric system depends on (a) medium interaction (water) with surface, (b) drug dissolution in water-filled pores governed by pore volume and drug solubility, (c) diffusion through water-filled channels adopting least resistant channels, (d) geometry and structure of pore network based on contributing and non-contributing porosity, (e) physical and chemical interactions, (f) partitioning of the diffusant within the matrix wall, and (g) low drug loading leading to recovery. All these factors behave independently, opposite, or synergistically to increase or retard the mass transfer, thereby leading to the dissolution of drug influencing drug release (8,27–29).
The present drug delivery system somewhat resembles the air-enclosed system, which showed a maximum floating time of 9 h depending upon condition of stomach and supine position of the subject (30). Based on the position of subject (lying during night), working against the principle of gastroretention, a floating study for 6 h was undertaken in pH 1.2 HCl IP medium maintaining sink conditions. All batches showed satisfactory floating characteristics during the time of experiment. This was followed by 3 h in pH 7.2 phosphate buffer IP taken as an appropriate dissolution medium to mimic increase in pH after floating. In our previous work, maximum impact of burst release occurred within 15 min followed by a very slow release or cutoff using basic medium for the same formulations (12). In present the formulation, being a multiparticulate system where there is no all-at-once emptying is noted, the analysis interval was set at 1 h after changing medium to normalize time for gastric emptying time and simultaneously the burst effect (13).
Overall drug release show different release profiles, ranging 13–24% at the end of 6 h in acidic medium followed by the burst release with range of 41–68% to the previously released drug. The end release ranging 69–94% at 9 h is shown in Fig. 6. Release profile of all batches at the end of 6 h decreased with increasing amount of the drug used for adsorption with maximum intradifference using 3 ml. Increased surface area phenomenon along with contribution of open pores seizing more dissolution medium due to low drug amounts resulted in higher release rates. Batches with high drug amounts showed the least release irrespective of solvent volume used, which were nearly half or more than the starting least amount. Factors for this phenomenon can be related to (a) restricted/blocked surface, leading to low pore volume restricting mass transfer, (b) effect of low drug solubility in medium, (c) formation of multiple dissolution fronts, (d) roughness of surface, and (e) moment of dissolved drug packets from within the isolated or interconnected pores toward the dissolution medium. The flow and interaction of the dissolution medium inside the porous material appears to be critical in observing mass transfer. No significant difference in drug release was noted considering the same drug amount at different solvent volumes, indicating similar amount of drug amount ready for mass transfer.
Batch 6, with least release of 12.77% in acidic medium, shows significance in formulating the ideal system for chronotherapy favoring low release during gastroretentive period designated as lag period (Fig. 6b). The attainment of such release without using any release modifiers or subjecting to coating by single or multiple polymers or formulating in the form of simple or press-coated tablets or in the form of modified capsule consisting of various excipients makes it exceptional and lucid to formulate.
Statistical interpretations for the drug release were calculated at various time intervals, which was preferred over percent release as presented in Table III. The values conferred for the drug release in acidic medium show the negative impact of drug coefficient (β) throughout the time period. Initially, the negative solvent effect (α), along with positive solvent-drug (αβ) interaction, was noted, which vanish afterwards probably due to the behavior/dissolution of the outermost drug layer. The negative values of drug–drug (β2) interaction up to half way indicate the dissolution of underlying drug layers from the surface influenced by adsorption pattern. These values suggest pronounced impact of drug coefficient at all levels irrespective of solvent volume.
Changing to phosphate buffer pH 7.2 IP initiated the sudden increase in release, with maximum burst release of 68.95% for batch 3. At the end of the experiment, batch 3 showed maximum release, while cut off was observed in some batches at 8-h mark. Sudden depletion of adsorbed drug clusters favors dominance of non-contributing porosity, thereby causing new boundary conditions. This leads to the exposure of underlying rough and hydrophobic nature of surface and pores to dissolution medium, which in turn assist in retarding/stopping the drug release. In other batches, slower drug release was observed after burst release was influenced by the continuity of adsorbed drug layer and its mass transfer momentum from inside to outside (29). Moving boundary conditions influencing geometry, described as Stephan moving problem (31,32), was perfectly observed in drug release while exposing porous carriers to basic medium alone than using acidic medium before it.
Statistically, drug as well as solvent–solvent (α2) interaction coefficient showed a positive influence on burst release fraction (Fig. 7a), while a positive influence of drug coefficient (β) was observed at 7 h. The same observation at the end of experiment shows the contributions from most coefficients conferring the positive influences of the selected variables, which worked ambiguously in acidic medium. Inference from this illustrates the influence of dissolution medium favoring difference in drug solubility as in Fig. 7b. The positive values of drug coefficient suggest its effect in basic medium only. These types of influences were similar to our previous study where only basic medium was used (12).
Batch 3 can be considered as optimized formulation for the purpose of chronotherapy, with 14.79% release in acidic medium followed by burst of 68.95%. In addition, batch 6 can be preferred as the subsequent choice with 12.77% release in acidic medium followed by 58.05% burst release.
Overall, the drug release ranging 8–15% was much lower unlike corresponding methanol batches in acidic medium. Burst effect ranges 31–70%, and the final release range 55–99% was observed with a positive difference in some batches, as seen in Fig. 8. Factors governing the release pattern were more or less the same, probably differing in magnitude.
The release in acidic medium showed a characteristic trend recording the least release of batches using medium level of drug (200 mg) irrespective of solvent volume used. Batch 8 recorded the least release of 8%, as in Fig. 8c. This release is much lesser than any batch even from the methanol batches. The least/less drug release can be related to the ever decreasing pore volume caused by adsorption of drug influenced by solvent property. This again supports the adsorption pattern of drug at meniscus of porous material using less polar solvent (24). The influence of the different solvent volume was not evident in acidic medium. Statistical interpretation show negative influence of drug (β) but a positive influence of drug–drug interaction (β2), as in Fig. 9a. This can be due to the development of various adsorption patterns in the form of stakes, which influence inter-pore connective influencing pore volume.
Burst release fraction, on changing to basic medium, observed increased release with increasing drug amount. It was twice or more for all solvent volumes used. The main feature, as shown by DCM, was the high burst release fraction for batch 3 at 70%. The less burst release in batch 1(lowest drug amount) can be due to fast exposure of underlying hydrophobic pores and surface, thereby retarding the drug release. These effects seem to be nullified with increasing amount of drug used for adsorption, where the adsorbed drug clusters act as the reservoirs for increased drug release. This difference can be summed up for the interaction of the nature of solvents and mediums affecting drug adsorption leading to the different rate mass transfer. Positive drug coefficient influence was observed statistically, which did not change when clubbed with total drug release for 7 h.
At the end of the experiment, batch 3 showed maximum release the same as that of methanol. Comparing with methanol batches, better drug release was observed using maximum drug amount, while a lesser drug release was calculated for other drug amounts. This can be due to variable mass transfer caused by boundary conditions of the adsorbed drug layer.
Statistically, at the end of the experiment, positive contributions from drug variables were prominent along with negative effect of solvent characteristic of solvent nature, as in Fig. 9b.
Overall, again, batch 3 can be considered as the most optimized formulation for effective floating pulsatile drug delivery with 13.48% release in acidic medium followed by burst of 70.91%. In addition, batch 9 can be preferred as the subsequent choice with 11.34% drug release in acidic medium and 66.95% as burst release.
Stability studies predict no significant changes in the drug release. Apart from this, DSC and XRD show the same results with marginal difference.
A lucid and simple floating pulsatile drug delivery system intended for chronotherapy was achieved using 32 factorial design for optimizing the formulation. Single-step adsorption techniques, unaided by additives, are promising enough for further evaluation of present and various other drug delivery systems. Using this process, four different batches with desired release parameters can be selected. BET and mercury data seem to behave differently in accordance with the principle of instrument working. Cross-sections predicted the inner core geometry of adsorbed porous carrier giving an insight about the possible drug release phenomenon. The modified geometry and network structure of pores in Accurel MP 1000® due to influence of selected variables govern the desired drug release pattern. The dissolution medium showing varying drug solubility shows multiple variations in pore volume and pore surface, therefore affecting porosity and permeability. Variable effect was dormant in acidic medium. This influenced the drug release rate process, which in turn is also dependent on static and changing adsorbing pattern of drugs. This work can be extended for time-scheduled drug release of drugs having low solubility, poor absorption, or degradation in lower gastrointestinal tract.
Praveen Sher is thankful to Dr. James R. Benson of Polygenetics, Inc. for his valuable advice during work. Praveen Sher and Atmaram Pawar are also thankful to UGC, India for providing major research project. Authors are thankful to Dr. Smita Mule (DSC), A.B. Gaikward (SEM), Baishakhi (AFM), and Supriya Palimkar for help in evaluation work. Authors also thank Membrana, Germany for providing a free gift sample of Accurel MP 1000®, particularly Ms. Claudia Gramann for her support.
Praveen Sher, Email: moc.liamg@neevarprehs.
Atmaram P. Pawar, Phone: +91-20-25437237, Fax: +91-20-25439383, Email: moc.liamffider@maramta_p.