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PM Physico-Chemical Characterization Determination of critical micelle concentration (CMC) Pyrene fluorescence for the determination of CMC and studying the interior of the PM One of the mostly used methods in PM characterization. For a pyrene molecule P; P > (excitation)> P* P* + P > P (excimer) Pe/Pm: measure of the ease of excimer (e) formation from the monomer (m) Excimer formation is function of microviscosity of the micelle core, Excimer formation is sensitive to pyrene concentration because it involves an interaction between two pyrene species. The emission spectrum of the pyrene monomer in the 350- to 420-nm region consists of five primary vibronic bands, usually designated as I1–I5, from shorter to longer wavelengths. band 1: shows significant intensity enhancements in polar environments; band 3: shows minimal variation in intensity with polarity changes. General Method Dissolve Pyrene in organic solvent, Add to concentration series of the polymer solution*, Evaporate the organic solvent, Measure fluorescent spectra using fluorescence spectrophotometer. * the final concentration of pyrene is in the 10−7 M range. CMC is determined using the ratio of peak intensities at 338 and 333 nm (I338/I333) from pyrene’s excitation spectra. A number of I338/I333 values is been obtained by varying the polymer concentration. When the polymer concentration is low, the I338/I333 value is the same as that of pyrene in water. When the polymer concentration increases, the red shift from 333 to 338 nm in the pyrene excitation spectra indicates the movement of pyrene into a more hydrophobic environment. Figure: Plot of the intensity ratio I338/I333 and I1/I3, which were obtained from the excitation and emission spectra, respectively, as a function of log C. The cmc was taken from the intersection of the horizontal line at low polymer concentrations with the tangent of the curve at high polymer concentrations. Measurement of lower critical solution temperature (LCST) or the cloud point Turbidity method using UV–vis Spectrophotometer by monitoring the transmittance at 500 nm at preset heating rate. LCST, why? Hydrophilic polymer + water hydrogen bond (exothermic), Hydrophobic polymer, Surrounded by water clusters (low entropy), At higher temperatures Release of water molecules (increase in S), Hyrophobic interaction (increase in S) polymer precipitation. Figure - Schematic representation of hydrophobic interaction Ref.: Physical Pharmacy Book J.X. Zhang et al. / Colloids and Surfaces B: Biointerfaces 43 (2005) 123–130 Particle size distribution of PM in aqueous solution of 0.5% at: (a) 25 ◦C; and (b) 45 ◦C. LCST=32.6 C. Self-assembly and thermally-induced change of a copolymer in aqueous solution. Note: the students get confused from the above figure, we should distinguish between increase in concentration and increase in temperature. Diameter changes of a PM as a function of temperature Micelle size: can be determined using light scattering methods, Micelle morphology: can be observed using transmission electron microscopy (TEM), …….. Micellar drug solubilization Surfactants and amphiphilic block copolymers can greatly affect the aqueous solubility of compounds by providing a hydrophobic reservoir where they can partition. Water solubility of some hydrophobic drugs was enhanced by a factor of 300 when incorporated into the core of some PM. The partitioning of some chemotherapeutic agents into the hydrophobic PM phase was highly favored with partition coefficients as high as 5.0x104. G. Gaucher et al. / Journal of Controlled Release 109 (2005) 169–188 Measurement of drug solubilized in the PM: By dissolving the lyophilized PM in organic solvent like DMSO. Drug concentration is then measured using suitable analytical method. Micelle stability Involves: Storage stability; Dilution stability; In vivo stability: Adsorption of Protein at PM surface >>PM clearance from the blood. OR PM-protein binding>>disrupt micelle cohesion>> premature drug release. Storage stability lyophilized drug-loaded PM are stored at specific temperature and humidity conditions and the samples are monitored for time-dependent changes in particle size and drug content during the storage period. Dilution stability The effect of dilution on the micelles can be studied by incubating the micelles in buffer solution at say 10-fold dilution at the required temperature for certain period. After filtration, the incubation solution is then analyzed for the presence of the drug. In vitro Drug release from the PM In vitro release profiles of the loaded drug from the PM is examined at specific conditions (vehicle, temperature, pH, ) using dialysis membrane of suitable MWCO. At predetermined time intervals, the amount of released drug is determined using suitable analytical method.