Designing sub-20 nm self-assembled nanocarriers for small molecule delivery: Interplay among structural geometry, assembly energetics, and cargo release kinetics.

Biological constraints in diseased tissues have motivated the need for small nanocarriers (10-30 nm) to achieve sufficient vascular extravasation and pervasive tumor penetration. This particle size limit is only an order of magnitude larger than small molecules, such that cargo loading is better described by co-assembly processes rather than simple encapsulation. Understanding the structural, kinetic, and energetic contributions of carrier-cargo co-assembly is thus critical to achieve molecular-level control towards predictable in vivo behavior. These interconnected set of properties were systematically examined using sub-20 nm self-assembled nanocarriers known as three-helix micelles (3HM). Both hydrophobicity and the "geometric packing parameter" dictate small molecule compatibility with 3HM's alkyl tail core. Planar obelisk-like apomorphine and doxorubicin (DOX) molecules intercalated well within the 3HM core and near the core-shell interface, forming an integral component to the co-assembly, as corroborated by small-angle X-ray and neutron-scattering structural studies. DOX promoted crystalline alkyl tail ordering, which significantly increased (+63%) the activation energy of 3HM subunit exchange. Subsequently, 3HM-DOX displayed slow-release kinetics (t1/2 = 40 h) at physiological temperatures, with ~50× greater cargo preference for the micelle core as described by two drug partitioning coefficients (micellar core/shell Kp1 ~ 24, and shell/bulk solvent Kp2 ~ 2). The geometric and energetic insights between nanocarrier and their small molecule cargos developed here will aid in broader efforts to deconvolute the interconnected properties of carrier-drug co-assemblies. Adding this knowledge to pharmacological and immunological explorations will expand our understanding of nanomedicine behavior throughout all the physical and in vivo processes they are intended to encounter.

Effect of alkyl length of peptide-polymer amphiphile on cargo encapsulation stability and pharmacokinetics of 3-helix micelles.

3-Helix micelles have demonstrated excellent in vitro and in vivo stability. Previous studies showed that the unique design of the peptide-polymer conjugate based on protein tertiary structure as the headgroup is the main design factor to achieve high kinetic stability. In this contribution, using amphiphiles with different alkyl tails, namely, C16 and C18, we quantified the effect of alkyl length on the stability of 3-helix micelles to delineate the contribution of the micellar core and shell on the micelle stability. Both amphiphiles form well-defined micelles, <20 nm in size, and show good stability, which can be attributed to the headgroup design. C18-micelles exhibit slightly higher kinetic stability in the presence of serum proteins at 37 °C, where the rate constant of subunit exchange is 0.20 h(-1) for C18-micelles vs 0.22 h(-1) for C16-micelles. The diffusion constant for drug release from C18-micelles is approximately half of that for C16-micelles. The differences between the two micelles are significantly more pronounced in terms of in vivo stability and extent of tumor accumulation. C18-micelles exhibit significantly longer blood circulation time of 29.5 h, whereas C16-micelles have a circulation time of 16.1 h. The extent of tumor accumulation at 48 h after injection is ∼43% higher for C18-micelles. The present studies underscore the importance of core composition on the biological behavior of 3-helix micelles. The quantification of the effect of this key design parameter on the stability of 3-helix micelles provides important guidelines for carrier selection and use in complex environment.

Distribution of nanoparticles throughout the cerebral cortex of rodents and non-human primates: Implications for gene and drug therapy.

When nanoparticles/proteins are infused into the brain, they are often transported to distal sites in a manner that is dependent both on the characteristics of the infusate and the region targeted. We have previously shown that adeno-associated virus (AAV) is disseminated within the brain by perivascular flow and also by axonal transport. Perivascular distribution usually does not depend strongly on the nature of the infusate. Many proteins, neutral liposomes and AAV particles distribute equally well by this route when infused under pressure into various parenchymal locations. In contrast, axonal transport requires receptor-mediated uptake of AAV by neurons and engagement with specific transport mechanisms previously demonstrated for other neurotropic viruses. Cerebrospinal fluid (CSF) represents yet another way in which brain anatomy may be exploited to distribute nanoparticles broadly in the central nervous system. In this study, we assessed the distribution and perivascular transport of nanoparticles of different sizes delivered into the parenchyma of rodents and CSF in non-human primates.

Evaluation of doxorubicin-loaded 3-helix micelles as nanocarriers.

Designing stable drug nanocarriers, 10-30 nm in size, would have significant impact on their transport in circulation, tumor penetration, and therapeutic efficacy. In the present study, biological properties of 3-helix micelles loaded with 8 wt % doxorubicin (DOX), ~15 nm in size, were characterized to validate their potential as a nanocarrier platform. DOX-loaded micelles exhibited high stability in terms of size and drug retention in concentrated protein environments similar to conditions after intravenous injections. DOX-loaded micelles were cytotoxic to PPC-1 and 4T1 cancer cells at levels comparable to free DOX. 3-Helix micelles can be disassembled by proteolytic degradation of peptide shell to enable drug release and clearance to minimize long-term accumulation. Local administration to normal rat striatum by convection enhanced delivery (CED) showed greater extent of drug distribution and reduced toxicity relative to free drug. Intravenous administration of DOX-loaded 3-helix micelles demonstrated improved tumor half-life and reduced toxicity to healthy tissues in comparison to free DOX. In vivo delivery of DOX-loaded 3-helix micelles through two different routes clearly indicates the potential of 3-helix micelles as safe and effective nanocarriers for cancer therapeutics.

3-Helix micelles stabilized by polymer springs.

Despite increasing demands to employ amphiphilic micelles as nanocarriers and nanoreactors, it remains a significant challenge to simultaneously reduce the particle size and enhance the particle stability. Complementary to covalent chemical bonding and attractive intermolecular interactions, entropic repulsion can be incorporated by rational design in the headgroup of an amphiphile to generate small micelles with enhanced stability. A new family of amphiphilic peptide-polymer conjugates is presented where the hydrophilic headgroup is composed of a 3-helix coiled coil with poly(ethylene glycol) attached to the exterior of the helix bundle. When micelles form, the PEG chains are confined in close proximity and are compressed to act as a spring to generate lateral pressure. The formation of 3-helix bundles determines the location and the directionalities of the force vector of each PEG elastic spring so as to slow down amphiphile desorption. Since each component of the amphiphile can be readily tailored, these micelles provide numerous opportunities to meet current demands for organic nanocarriers with tunable stability in life science and energy science. Furthermore, present studies open new avenues to use energy arising from entropic polymer chain deformation to self-assemble energetically stable, single nanoscopic objects, much like repulsion that stabilizes bulk assemblies of colloidal particles.

Long-circulating 15 nm micelles based on amphiphilic 3-helix peptide-PEG conjugates.

Generating stable, multifunctional organic nanocarriers will have a significant impact on drug formulation. However, it remains a significant challenge to generate organic nanocarriers with a long circulation half-life, effective tumor penetration, and efficient clearance of metabolites. We have advanced this goal by designing a new family of amphiphiles based on coiled-coil 3-helix bundle forming peptide-poly(ethylene glycol) conjugates. The amphiphiles self-assemble into monodisperse micellar nanoparticles, 15 nm in diameter. Using the 3-helix micelles, a drug loading of ∼8 wt % was obtained using doxorubicin and the micelles showed minimal cargo leakage after 12 h of incubation with serum proteins at 37 °C. In vivo pharmacokinetics studies using positron emission tomography showed a circulation half-life of 29.5 h and minimal accumulation in the liver and spleen. The demonstrated strategy, by incorporating unique protein tertiary structure in the headgroup of an amphiphile, opens new avenues to generate organic nanoparticles with tunable stability, ligand clustering, and controlled disassembly to meet current demands in nanomedicine.

Amphiphilic peptide-polymer conjugates with side-conjugation.

Polymers conjugated to the exterior of a protein mediate its interactions with surroundings, enhance its processability and can be used to direct its macroscopic assemblies. Most studies to date have focused on peptide-polymer conjugates based on hydrophilic polymers. Engineering amphiphilicity into protein motifs by covalently linking hydrophobic polymers has the potential to interface peptides and proteins with synthetic polymers, organic solvents, and lipids to fabricate functional hybrid materials. Here, we synthesized amphiphilic peptide-polymer conjugates in which a hydrophobic polymer is conjugated to the exterior of a heme-binding four-helix bundle and systematically investigated the effects of the hydrophobicity of the conjugated polymer on the peptide structure and the integrity of the heme-binding pocket. In aqueous solution with surfactants present, the side-conjugated hydrophobic polymers unfold peptides and may induce an α-helix to ß-sheet conformational transition. These effects decrease as the polymer becomes less hydrophobic and directly correlate with the polymer hydrophobicity. Upon adding organic solvent to solubilize the hydrophobic polymers, however, the deleterious effects of hydrophobic polymers on the peptide structures can be eliminated. Present studies demonstrate that protein structure is sensitive to the local environment. It is feasible to dissolve amphiphilic peptide-polymer conjugates in organic solvents to enhance their solution processability while maintaining the protein structures.

Electrospraying: a facile technique for synthesis of chitosan-based micro/nanospheres for drug delivery applications.

Electrospraying is a novel technique for the generation of micro/nanospheres for biomedical applications. Apart from being a high yield technique; electrospraying has an added advantage of not making use of an external dispersion/emulsion phase which often involves ingredients that are undesirable for biomedical applications. In this study, we report the use of electrospraying for the synthesis of chitosan micro/nanospheres. The focus was to optimize the fabrication parameters involved in electrospraying for reproducible synthesis of chitosan based micro/nanospheres and to study their potential as delivery vehicles for bioactive agents. The influence of the following was studied (i) electrospraying voltage, (ii) needle gauge, (iii) concentration of chitosan solution, (iv) concentration of acetic acid solution, and (v) electrospraying distance. SEM analysis demonstrated that microspheres of less than 1 mum were obtained when chitosan concentration was 2% dissolved in 90% acetic acid. The working distance and needle gauge that yielded favorable results were 7 cm and 26 g, respectively. Average particle size of ampicillin loaded chitosan micro/nanospheres was 520 nm with zeta potential of +28.2 mV and encapsulation efficiency of 80.4%. The particles were characterized for drug release kinetics and results demonstrated an initial burst release followed by a sustained release over a period of 120 h. Further, antibacterial activity of drug loaded micro/nanospheres demonstrated that the encapsulated drug was in its active form postexposure to high voltage during electrospraying. This study indicates that electrospraying is a facile technique for the synthesis of chitosan micro/nanospheres for drug delivery applications.