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 1000-folds 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 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

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 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.

Dialysis method: the copolymer mixture can be dialyzed against water 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-water-miscible 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

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.

Dissolve Pyrene in organic solvent, 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.

Ref.: Physical Pharmacy Book 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 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.