Sergey Kazakov

Liposome-Nanogel Structures for Future Pharmaceutical Applications

Nanoparticles have been extensively studied as drug delivery systems. In this review, we focus on a relatively new type of nanoparticles--lipobeads--a liposome-hydrogel assembly as a novel drug delivery system. An appropriate assemblage of spherical hydrogel particles and liposomes combines the properties of both classes of materials and may find a variety of biomedical applications. The bi-compartmental structure of lipobeads is a natural configuration. Thus, the technology of their preparation can be a key step of designing more stable and effective vaccines. Biocompatibility and stability, ability to deliver a broad range of bioactive molecules, environmental responsiveness of both inner nanogel core and external lipid bilayer, and individual specificity of both compartments make the liposome-nanogel design a versatile drug delivery system relevant for all known drug administration routes and suitable for different diseases with possibility of efficient targeting to different organs. New findings on reversible and irreversible aggregation of lipobeads can lead to novel combined drug delivery systems regarding lipobeads as multipurpose containers. The research on hydrogel-liposome submicrometer structures has just begun and fundamental studies on interactions between hydrogels and liposomes are in demand.

Physicochemical Characterization of Natural Ionic Microreservoirs: Bacillus subtilis Dormant Spores

The kinetics of proton exchange between dormant spores and aqueous environment was examined by time-resolved micropotentiometry, the method we recently introduced for hydrogel particles of micro- and nanometer diameter (J. Phys. Chem. B 2006, 110, 15107). In this work, the method was applied to the suspensions of dormant Bacillus subtilis spore of different concentrations to show that proton uptake kinetics was a multistep process involving a number of successively approximately 10-fold slower steps of proton penetration into the bulk and their binding to the ionizable groups within different layers of a spore structure. By analyzing the proton equilibrium binding to ionizable groups inside a spore, it was shown that each Bacillus subtilis spore behaves like almost infinite ionic reservoir capable of accumulating billions of protons (N approximately 2 x 10(10) per spore). The obtained pK(a) value of 4.7 for the spores studied is the first quantitative indication on carboxyl groups as the major ionizable groups fixed in a spore matrix. In general, proton equilibrium binding within the spore matrix obeys the fundamental law of the Langmuir isotherm. The proton binding to the ionizable groups slows down the free proton diffusion within a spore, but this effect is substantially weakened by increasing the initial concentration of protons added. On the basis of the diffusion time analysis, it was found that the effective diffusion coefficient for hydrogen ions within the spore core can be up to 3 orders of magnitude lower than that within the coats and cortex. We speculate that the spore inner membrane which separates core from cortex and coats in a dormant spore is a major permeability barrier for protons to penetrate into a lockbox of the genetic information (core).

Liposome-Nanogel Structures for Future Pharmaceutical Applications: An Updated Review

Nature uses combination of lipid bilayers and cross-linked macromolecular networks to achieve workability, multifunctionality, and dynamism in living cells of different types. Despite the concept of liposome-nanogel structures (lipobeads) is known for about 30 years, lipobead-based drug delivery systems are still largely experimental. The data available on nano- and giant lipobeads are reviewed to demonstrate technological achievability of lipobeads and to support the expectations that additional expenses on their production will be reimbursed by the potential advantages of their use. Indeed, lipobeads exhibit the properties attractive for the next generation of drug delivery systems: (i) retaining all the important benefits of polymeric and liposomal drug carriers, the hydrogel core brings mechanical stability and environmental responsiveness to the formulation in one construct, (ii) lipobeaddelivered combination therapy shows no toxicity on intravenously administered mice, accumulation of drug-loaded lipobeads both in the area surrounding tumor and within the tumor itself outside the vasculature, high therapeutic activity at the targeted site, and drastically increased survival, (iii) bipartite structure of lipobeads can provide a number of novel and unique options (e.g., consecutive multistep triggering and combined drug delivery systems). In addition, some ideas on the conceptually new drug delivery systems, new mechanisms of lipobead internalization into the cell and new schemes of drug release regulated by specific signaling are discussed.

Bioactive Flavonoids and Catechols as Hif1 and Nrf2 Protein Stabilizers - Implications for Parkinson’s Disease

Flavonoids are known to trigger the intrinsic genetic adaptive programs to hypoxic or oxidative stress via estrogen receptor engagement or upstream kinase activation. To reveal specific structural requirements for direct stabilization of the transcription factors responsible for triggering the antihypoxic and antioxidant programs, we studied flavones, isoflavones and catechols including dihydroxybenzoate, didox, levodopa, and nordihydroguaiaretic acid (NDGA), using novel luciferase-based reporters specific for the first step in HIF1 or Nrf2 protein stabilization. Distinct structural requirements for either transcription factor stabilization have been found: as expected, these requirements for activation of HIF ODD-luc reporter correlate with in silico binding to HIF prolyl hydroxylase. By contrast, stabilization of Nrf2 requires the presence of 3,4-dihydroxy- (catechol) groups. Thus, only some but not all flavonoids are direct activators of the hypoxic and antioxidant genetic programs. NDGA from the Creosote bush resembles the best flavonoids in their ability to directly stabilize HIF1 and Nrf2 and is superior with respect to LOX inhibition thus favoring this compound over others. Given much higher bioavailability and stability of NDGA than any flavonoid, NDGA has been tested in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-animal model of Parkinson’s Disease and demonstrated neuroprotective effects.

Nanometer scale ionic reservoir based on ion-responsive hydrogels

The applicability of the concept of ionic reservoir for the description of hydrogel behavior was demonstrated by potentiometric titration of poly(N-isopropylacrylamide-co-1- vinylimidazole) hydrogel suspension. Four different regions of pH-changes of the microgel suspensions were identified on the titration curve in comparison with pure water. Particularly, at 10.5>pH>6.5 a hydrogel accumulates or releases H+ and Cl- ions without significant swelling/deswelling whereas at 6.5>pH>4 the storage of the ions occurs both due to their binding with ionizable groups on polymer network and due to strong swelling. The mechanical response of hydrogel (swelling/deswelling) is assumed to be a faster process than the electrochemical response (equilibration of ion concentrations interior and exterior to the hydrogel). The size of hydrogel spheres should be diminished to fasten an ionic reservoir response of the hydrogel. A novel protocol for preparation of polymer hydrogel spherical particles on a nanometer scale (nanogels) has been developed. Temperature- and pH-sensitive nanogels were detected and characterized by the dynamic light scattering technique and atomic force microscopy. Ptoentiometric titration of the obtained nanogels shows that the decrease in the ionic reservoir size gains the efficiency and, presumably, the rate of the electrochemical response. These findings indicate the necessity of time-resolved pH-measurements of the hydrogel suspensions for the characterization of the rate of the solute diffusion through the gel/water surface.

Interaction of modified liposomes with Bacillus spores

The interaction between liposomes modified with a particular peptide sequence and Bacillus subtilis spores was experimentally observed as (1) an increase in the average diameter of spore-related particles, and (2) the formation of dense and structured shells around the spores at higher concentrations of liposomes.

Evanescent wave spectroscopy for studying swelling/de-swelling kinetics of soft matter

Optical fibers connected to a spectrophotometer are shown to be analytical devices for recording the kinetics of structural changes within soft materials deposited on the side surface of the fiber core. A portion of electromagnetic radiation propagating inside an optical fiber penetrates out of its core (evanescent field) and interacts with molecules located within the depth of penetration (∼λ/10). It can be absorbed, scattered or can excite molecules around the fiber core surface. To prove the concept, a cylindrical hydrogel film [poly-N-(isopropylacrylamide), PNIPA] was polymerized by an evanescent wave around the fiber core immerged into the hydrogel forming solution. The integral intensity of light passing through the fiber core covered with the dried PNIPA hydrogel film was recorded with time during its hydration to reveal tiny features of swelling kinetics. The volume transition temperature (∼32 °C) was determined for the PNIPA hydrogel by measuring integral intensity and spectral composition of light passing through the combined gel-on-fiber core system at different temperatures. This value agrees well with the one known from the literature. Above this temperature, the PNIPA hydrogel shrank absorbing/scattering the light in different spectral ranges with a temperature increase. The results are of great importance for practical applications such as the time-resolved spectroscopy of structural transformations within not only synthetic, but also natural soft materials (cells, organelles, microorganisms, etc.).

Hydrogel Micro-/Nanosphere Coated by a Lipid Bilayer: Preparation and Microscopic Probing

The result of polymeric nanogels and lipid vesicles interaction—lipobeads—can be considered as multipurpose containers for future therapeutic applications, such as targeted anticancer chemotherapy with superior tumor response and minimum side effects. In this work, micrometer sized lipobeads were synthesized by two methods: (i) mixing separately prepared microgels made of poly(N-isopropylacrylamide) (PNIPA) and phospholipid vesicles of micrometer or nanometer size and (ii) polymerization within the lipid vesicles. For the first time, a high vacuum scanning electron microscopy was shown to be suitable for a quick validation of the structural organization of wet lipobeads and their constituents without special sample preparation. In particular, the structural difference of microgels prepared by thermal and UV-polymerization in different solvents was revealed and three types of giant liposomes were recognized under high vacuum in conjunction with their size, composition, and method of preparation. Importantly, the substructure of the hydrogel core and multi- and unilamellar constructions of the peripheral lipid part were explicitly distinguished on the SEM images of lipobeads, justifying the spontaneous formation of a lipid bilayer on the surface of microgels and evidencing an energetically favorable structural organization of the hydrogel/lipid bilayer assembly. This key property can facilitate lipobeads’ preparation and decrease technological expenses on their scaled production. The comparison of the SEM imaging with the scanning confocal and atomic force microscopies data are also presented in the discussion.