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PART 1 Selected Topics In Physical Pharmacy A. Physico-Chemical Characterization of Some Surfactant Containing Systems 1. Polymeric Micelles • PM are under investigation mainly as carriers for anti-tumor drugs, contrast agents for diagnostic imaging, enzymes, in gene delivery, … • Polymeric micelles, PM, belong to the class of “nanomedicine”, • In drug delivery, PM are classified under the “nanocarriers”, • Table: Summary of different nanotherapeutic technologies proposed for cancer therapy • General Structure of the PM Schematic illustration of the core-shell architecture of a polymer micelle and its dimensions General Structure of the PM Ref.: Pharmacology & Therapeutics 112 (2006) 630–648 Advantages of PM as nanodrug carriers • Drug protection Drugs incorporated in the core of the PM are protected from harsh biological environments (e.g. low pH and hydrolytic enzymes), • Drug Solubilization Hdrophobic drugs can be imbibed into the hydrophobic inner core of the PM, significantly improving their water solubility. • Stability (low CMC & long circulation period) PM have low CMC or CAC “The lower is the CMC value of a given amphiphilic polymer, the more stable are micelles even at low concentration of an amphiphile in the medium. This is especially important from the pharmacological point of view, since upon the dilution with the large volume of the blood only micelles with low CMC value still exist, while micelles with high CMC value dissociate into unimers and their content precipitates in the blood.” V.P. Torchilin / Advanced Drug Delivery Reviews 54 (2002) 235 –252 • The critical association concentration, CAC defines a threshold concentration for PM assembly. • Polymeric micelles may not necessarily dissociate immediately after extreme dilution following intravenous injection into the body because they have a remarkably low CAC, (10–6 – 10–7 M), which is 1000folds lower than that of surfactant micelles. Stability- cont. PM dissociation is kinetically slow. This property allows the micelles to circulate in the bloodstream until accumulation at target tissues. For example: some PEG-b-PDLLA PM micelles showed a remarkably prolonged blood circulation (t1/2 about 18 hr) after intravenous administration, and maintained 25% of the injected dose in the circulation at 24-hr post-injection. Also, the presence of hydrophilic polymers on the surfaces of PM is known to hinder protein adsorption and opsonization of the particles by the reticuloendothelial system (RES) which is responsible for engulfing and clearing old cells, miscellaneous cellular debris, foreign substances, and pathogens from the bloodstream. • Safety of the PM The constituent block copolymers might be finally excreted into the urine due to their molecular weight being lower than the threshold of glomerular filtration, suggesting the safety of polymeric micelles with a low risk of chronic accumulation in the body. • Targetability In cancer therapy and diagnostic imaging, PM have special importance, why ? PM physico-chemical properties. PM (and other nanocarriers) size allows for passive tumor targeting (EPR). “Tumor blood vessels are generally characterized by abnormalities accompanied by decreases lymphatic drainage and renders the vessels permeable to macromolecules. Because of the decreased lymphatic drainage, the permeant macromolecules are not removed efficiently, and are thus retained in the tumor. This passive targeting phenomenon, has been called the ‘‘enhanced permeation and retention (EPR) effect’’. EPR effect results in passive accumulation of macromolecules and nanosized particulates (e.g. polymer conjugates, polymeric micelles, dendrimers, and liposomes) in solid tumor tissues, increasing the therapeutic index while decreasing side effects.” J.H. Park et al. / Prog. Polym. Sci. 33 (2008) 113–137 • The PM structure allows for active targeting. Specific ligands such as antibodies can be attached to the hydrophilic block polymer. • Figure: Multicomponent targeting strategies. Nanoparticles extravasate into the tumour stroma through the fenestrations of the angiogenic vasculature, demonstrating targeting by enhanced permeation and retention. • The particles carry multiple antibodies, which further target them to epitopes on cancer cells, and direct antitumour action. • Nanoparticles are activated and release their cytotoxic action when irradiated by external energy. The red blood cells are not shown to scale; the volume occupied by a red blood cell would suffice to host 1–10 million nanoparticles of 10 nm diameter. NATURE REVIEWS | CANCER VOLUME 5, 2005, 161-171 PART 2 Polymeric Micelles • PM are formed from the association of block copolymers. • Block copolymers micelles have the capacity to increase the solubility of hydrophobic molecules due to their unique structural composition, which is characterized by a hydrophobic core sterically stabilized by a hydrophilic shell. The hydrophobic core serves as a reservoir in which the drug molecules can be incorporated by means of chemical, physical or electrostatic interactions, depending on their physicochemical properties. Block vs. Random Diblock vs Multiblock natural (dextran) and synthetic (polystyrene) polymeric micelle. Daniel Taton and co-workers at the University of Bordeaux, France, 2007 According to the type of intermolecular forces driving the micelle formation, PM are classified as: • amphiphilic micelles; formed by non polar hydrophobic interactions, • polyion complex micelles, PICM; resulting from electrostatic interactions, • micelles formed by metal complexation. Non polar hydrophobic interactions Micellization is driven by a gain in entropy of the solvent molecules as the hydrophobic components withdraw from the aqueous media. Examples: Polymers contain • polyester as Poly(lactic acid) (PLA),… • poly(amino acid) PAA derivative as Poly(aspartic acid) (PAsp),… (neutral or conjugated to hydrophobic group). ,….... • Polyethers as the poloxamer family { (poly(ethylene glycol)-b-poly(propylene oxide)-bpoly( ethylene glycol)) (PEG-b-PPO-b-PEG) All are biocompatible and biodegradable approved by the FDA for biomedical applications in humans. Electrostatic interactions The self-assembly of PICM, proceeds through the electrostatic interaction between polycations and polyanions leading to neutralization of oppositely charged polyions. The presence of the polymer hydrophilic segment prevents precipitation. Examples: • Polymers having protonated amines at physiological pH like poly(ethyleneimine) (PEI),….. Micelle Preparation Among the methods used in PM preparation are 1. Direct dissolution method Suitable for moderately hydrophobic copolymers, such as poloxamers, and for PICM. dissolving the block copolymer along with the drug in an aqueous solvent. The copolymer and drug are dissolved separately in an injectable aqueous vehicle. Micelle formation is induced by combining the two solutions to appropriate drug–polymer /charge ratios. 2. Indirect method using organic solvent Applies to amphiphilic copolymers which are not readily soluble in water. Organic solvent common to both the copolymer and the drug is used. (such as dimethylsulfoxide, acetone,…. The mechanism by which micelle formation is induced depends on the solvent-removal procedure. a. Dialysis method: the copolymer mixture can be dialyzed against water. Slow removal of the organic phase triggers micellization. b. Solution-casting method: evaporation of the organic phase to yield a polymeric film where polymer–drug interactions are favored. Rehydration of the film with a heated aqueous solvent produces drug-loaded micelles. c. Emulsion (O/W) method using non-watermiscible organic solvent like dichloromethane. d. Freeze drying method: one-step procedure based on the dissolution of both the polymer and the drug in a water/tert-butanol (TBA) mixture with subsequent lyophilization of the solvents. Drug-loaded micelles are formed spontaneously upon reconstitution of the freeze-dried polymer–drug cake in an injectable vehicle. Common drug-loading procedures: (A) simple equilibrium, (B) dialysis, (C) O/W emulsion, (D) solution casting, and (E) freeze-drying. Journal of Controlled Release 109 (2005) 169–188 Ref.: © 2004 IUPAC, Pure and Applied Chemistry 76, 1321–1335 PART 3 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. PART 4 Evaluation of Polymeric Micelles from Brush Polymer with Poly(ε-caprolactone)-bPoly(ethylene glycol) Side Chains as Drug Carrier Biomacromolecules 2009, 10, 2169–2174 Introduction Polymeric micelles self-assembled from amphiphilic copolymers have attracted significant attention in the fields of biomedical applications. They have been developed as delivery systems of drug as well as contrast agents in diagnostic imaging applications. In an aqueous environment, the hydrophobic component of the copolymer is expected to segregate into the core of the micelles, while the hydrophilic component forms the corona or outer shell. The hydrophobic micelle core serves as a microenvironment for incorporation of various therapeutic compounds, while the corona can act as a stabilizing interface between the hydrophobic core and the external medium. Although various micelles have been developed for in vitro and in vivo studies and applications, the majority of works have been focused on micelles formed by intermolecular aggregation of amphiphilic linear block copolymers. Micelles from polymers with other architectures as drug delivery vectors are less studied, especially brush-like macromolecules. However, it has been reported that the architecture of polymer may determine the static and dynamic stability, morphology, size and size distribution of the micelles, and further affect the performance of micelles including drug loading and release rate, even in vivo circulation and distribution. Cylindrical brush polymers have recently attracted considerable attention from polymeric chemists. In this work, we synthesized cylindrical brush polymers PHEMA-g-(PCL-b-PEG) with poly(2-hydroxyethyl methacrylate) (PHEMA) as the backbone and poly(εcaprolactone)-bpoly( ethylene glycol) (PCL-b-PEG) amphiphilic block copolymers as the side chains. The micelle formation of these polymers was studied, and doxorubicin (DOX) was used to evaluate the drug loading and release behavior from the micelles. The internalization and cytotoxicity of drug-loaded micelles against A549 human lung carcinoma cells were also investigated. Experiments and Results Experimental Materials Different polymers and reagents. Methods Preparation of Micelles Micelles were prepared by a dialysis method. Briefly, PHEMA-g-(PCL-b-PEG) or PCL-b-PEG (10 mg) was dissolved in 2 mL of dimethyl sulfoxide (DMSO) and stirred for 2 h at room temperature. Then, the polymer solution was added dropwise into 5 mL of ultrapurified water under vigorous stirring. Two hours later, the solution was transferred into dialysis membrane tubing and dialyzed for 24 h against water to remove the organic solvent. Transmission Electron Microscopy (TEM) TEM was performed on a transmission electron microscope for the measurement of micelle morphology and dimensions. Result The formation of micellar nanoparticles was confirmed by TEM observations. Formed micelles are well-dispersed and display spherical morphology. They are relatively uniform in size and the average diameters are approximately 45 nm. Fluorescence Measurements. The fluorescent probe method was employed to determine the critical micellization concentration (CMC) of the micelles as followed. A predetermined amount of pyrene solution in acetone was added into a series of volumetric flasks, and the acetone was then evaporated completely. A series of copolymer solutions at different concentrations ranging from 1.0 × 10-5 to 1.0 mg mL1 were added to the flasks, while the concentration of pyrene in each flask was fixed at a constant value (6.0 × 10-7 mol L-1). The excitation spectra were recorded at 25 °C at λem of 390 nm. Result With increasing the polymer concentration, several changes in the fluorescence spectra of pyrene were observed. The intensity ratio of bands at 338 and 335 nm (I338/ I335) was calculated and plotted against the polymer concentrations, giving a sigmoid curve as shown in Figure 3. I338/I335 remained nearly unchanged at low polymer concentrations and increased dramatically when the polymer concentration reached a certain value, exhibiting the characteristic of pyrene entirely in a hydrophobic environment, which is actually a reflection of micelle formation. Preparation of DOX-Loaded Micelles DOX-loaded micelles were prepared similarly to the blank micelles. Briefly, 10 mg of each polymer was dissolved in 2 mL of DMSO, followed by adding a predetermined amount of DOX· HCl and two molar equivalents of triethylamine (TEA) and stirred at room temperature for 2 h. Then, the mixed solution was added dropwise to 5 mL of water. After being stirred for an additional 2 h, the solution was dialyzed against water for 24 h. The solution was further centrifuged at 3000 g for 3 min to remove free DOX. Determination of DOX Loading Content (DLC) and Loading Efficiency (DLE) To measure the amount of DOX trapped in the micelles, an aliquot of the DOX-loaded micelle solution was lyophilized and dissolved in DMSO. The concentration of DOX was measured by HPLC analyses as described below. The percentages of DLC and DLE were calculated according to the following equations: Results Size and size distributions of DOX-loaded micelles by DLS measurements revealed that encapsulation of DOX into micelles increased the diameters of micelles, as summarized in Table 3. In vitro Drug Release DOX-loaded micelle solution was diluted to 1 mg mL-1 in phosphate buffered saline, and transferred into a dialysis membrane tubing. The tubing was immersed in 20 mL of PBS and shaken at 37 °C. At a predetermined time interval, the external buffer of the tubing was collected, and it was replaced by fresh PBS. The concentration of DOX was determined by HPLC analyses with a fluorescence detector. Results DOX release kinetics was indeed affected by polymer architectures. Cell Culture A549 human lung carcinoma cells (ATCCs) were cultured in suitable conditions. Confocal Laser Scanning Microscopy (CLSM) A549 cells were incubated at 37 °C. Twenty-four hours later, free DOX (6 µM) and DOX-loaded micelles (final DOX concentration at 6 µM) were added to the cells, and the cells were incubated for 2 h. The cells were then observed with a Zeiss LSM510 Laser Confocal Scanning Microscope imaging system. Results In the Figure, cells incubated with free DOX showed strong fluorescence in cell nuclei, while very weak fluorescence was observed in cytoplasm, indicating DOX molecules entered the cells and rapidly accumulated in the nuclei. On the contrary, after 2 h incubation of DOX-loaded brush copolymer micelles, intense DOX fluorescence was observed in the cytoplasm rather than in cell nuclei. It implies that DOX loaded brush copolymer micelles can be effectively internalized by A549 cells. Cytotoxicity Study Cytotoxicity of DOX-loaded micelles and free DOX were measured against A549 cells where the relative cell viability was calculated. Results As shown in Figure 6, DOX-loaded brush polymer micelles exhibited relatively lower cytotoxicity to A549 cells when compared with free DOX at the same dose. Conclusions The brush polymers self-assembled into spherical micelles with the diameter less than 100 nm in aqueous solution. The brush polymer micelles showed enhanced aqueous stability than linear PCL-b-PEG diblock copolymer with similar structure to the side chains as determined by fluorescent probe method. When used as drug delivery carriers, the brush polymer micelles exhibited higher DOX loading capacity than the micelles from linear PCLb- PEG, and the burst drug release in the initial period was significantly suppressed. The micelles can be effectively internalized by A549 cells and slowly released the encapsulated drug molecules. They showed less potent but effective cell proliferation inhibition compared to free DOX. With these advantages, the brush polymer micelles are potential carriers for efficient drug delivery.