Microfluidic Assembly To Synthesize Dual Enzyme/Oxidation-Responsive Polyester-Based Nanoparticulates with Controlled Sizes for Drug Delivery.

Controlling the size and narrow size distribution of polymer-based nanocarriers for targeted drug delivery is an important parameter that significantly influences their colloidal stability, biodistribution, and targeting ability. Herein, we report a high-throughput microfluidic process to fabricate colloidally stable aqueous nanoparticulate colloids with tunable sizes at 50-150 nm and narrow size distribution. The nanoparticulates are designed with different molecular weight polyesters having both ester bonds (responsive to esterase) and sulfide linkages (to oxidative reaction) on the backbones, thus exhibiting dual esterase/oxidation responses, causing the destabilization of the nanoparticulates to lead to the controlled release of encapsulated therapeutics. The systematic investigation on both microfluidic and formulation parameters enables to control their properties as allowing for decreasing nanoparticulate sizes as well as improving colloidal stability and cytotoxicity. Further to such control over smaller size and narrow size distribution, dual stimuli-responsive degradation and excellent cellular uptake could suggest that the microfluidic nanoparticulates stabilized with polymeric stabilizers could offer the versatility toward dual smart drug delivery exhibiting enhanced release kinetics.

Self-Associating Poly(ethylene oxide)-block-poly(α-carboxyl-ε-caprolactone) Drug Conjugates for the Delivery of STAT3 Inhibitor JSI-124: Potential Application in Cancer Immunotherapy.

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) in tumor cells and tumor associated dendritic cells (DCs) plays a major role in the progression of cancer. JSI-124 (cucurbitacin I) is a potent inhibitor of STAT3; however, its poor solubility and nonspecificity limit its effectiveness in cancer immunotherapy. In order to achieve a nanocarrier for solubilization and passive targeting of JSI-124 to tumor cells and tumor associated DCs, the drug was chemically conjugated to pendent COOH groups of self-associating poly(ethylene oxide)-block-poly(α-carboxylate-ε-caprolactone) (PEO-b-PCCL). Developed PEO-b-P(CL-JSI-124) conjugates self-assembled to polymeric micelles of 40 nm size range with negligible drug release under physiological mimicking conditions. The conjugation of JSI-124 to PEO-b-PCCL was confirmed by 1H NMR, thin layer chromatography (TLC), and HPLC with a conjugation of 8.9% w/w of the polymer. As expected, JSI-124 nanoconjugates showed lower potency in p-STAT3 inhibition and direct anticancer activity in B16-F10 melanoma cells. Interestingly, JSI-124 nanoconjugates were more powerful than free drug in reducing the level of p-STAT3 in tumor exposed bone marrow derived dendritic cells (BMDCs). The JSI-124 nanoconjugates were also significantly more active than free drug in reversing the immunosuppressive effect of B16-F10 tumor and led to significantly better phenotypical and functional stimulation of tumor exposed immature BMDCs in the presence of immune adjuvants like LPS and CpG. Our findings points to great promise for PEO-b-P(CL-JSI-124) micelles for modulation of immunosuppressive microenvironment in melanoma tumors, implicating application of this strategy in cancer immunotherapy.

Microfluidic Production and Application of Lipid Nanoparticles for Nucleic Acid Transfection.

Lipid nanoparticles (LNPs) are established in the biopharmaceutical industry for efficient encapsulation and cytosolic delivery of nucleic acids for potential therapeutics, with several formulations in clinical trials. The advantages of LNPs can also be applied in basic research and discovery with a microfluidic method of preparation now commercially available that allows preparations to be scaled down to quantities appropriate for cell culture. These preparations conserve expensive nucleic acids while maintaining the particle characteristics that have made LNPs successful in later stages of genetic medicine development. Additionally, this method and the resulting LNPs are seamlessly scalable to quantities appropriate for in vivo models and development of nucleic acid therapeutics.The present work describes the methodology for preparing LNPs loaded with siRNA, mRNA or plasmids using a commercially available microfluidic instrument and an accompanying transfection kit. Guidelines for application to cultured cells in a well-plate format are also provided.

Micellar nano-carriers for the delivery of STAT3 dimerization inhibitors to melanoma.

The objective of this research was to develop polymeric micellar formulations of inhibitors of signal transducer and activator of transcription 3 (STAT3) dimerization, i.e., S3I-1757 and S3I-201, and evaluate the activity of successful formulations in B16-F10 melanoma, a STAT3 hyperactive cancer model, in vitro and in vivo. STAT3 inhibitory agents were encapsulated in methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) (PEO114-b-PCL22) and methoxy poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) (PEO114-b-PBCL20) micelles using co-solvent evaporation. Polymeric micelles of S3I-1757 showed high encapsulation efficiency (>88%), slow release profile (<32% release in 24 h) under physiological conditions, and a desirable average diameter for tumor targeting (33-54 nm). The same formulations showed low encapsulation efficiencies and rapid drug release for S3I-201. Further studies evidenced the delivery of functional S3I-1757 by polymeric micelles to B16-F10 melanoma cells, leading to a dose-dependent inhibition of cell growth and vascular endothelial growth factor (VEGF) production comparable with that of free drug. Encapsulation of S3I-1757 in polymeric micelles significantly reduced its cytotoxicity in normal bone marrow-derived dendritic cells (DCs). Micelles of S3I-1757 were able to significantly improve the function of B16-F10 tumor-exposed immunosuppressed DCs in the production of IL-12, an indication for functionality in the induction of cell-mediated immune response. In a B16-F10 melanoma mouse model, S3I-1757 micelles inhibited tumor growth and enhanced the survival of tumor-bearing mice more than free S3I-1757. Our findings show that both PCL- and PBCL-based polymeric micelles have potential for the solubilization and delivery of S3I-1757, a potent STAT3 inhibitory agent.

Traceable PEO-poly(ester) micelles for breast cancer targeting: The effect of core structure and targeting peptide on micellar tumor accumulation.

Traceable poly(ethylene oxide)-poly(ester) micelles were developed through chemical conjugation of a near-infrared (NIR) dye to the poly(ester) end by click chemistry. This strategy was tried for micelles with poly(ε-caprolactone) (PCL) or poly(α-benzyl carboxylate-ε-caprolactone) (PBCL) cores. The surface of both micelles was also modified with the breast cancer targeting peptide, P18-4. The results showed the positive contribution of PBCL over PCL core on micellar thermodynamic and kinetic stability as well as accumulation in primary orthotopic MDA-MB-231 tumors within 4-96 h following intravenous administration in mice. This was in contrast to in vitro studies where better uptake of PEO-PCL versus PEO-PBCL micelles by MDA-MB-231 cells was observed. The presence of P18-4 enhanced the in vitro cell uptake and homing of both polymeric micelles in breast tumors, but only at early time points. In conclusion, the use of developed NIR labeling technique provided means for following the fate of PEO-poly(ester) based nano-carriers in live animals. Our results showed micellar stabilization through the use of PBCL over PCL cores, to have a more significant effect in enhancing the level and duration of nano-carrier accumulation in primary breast tumors than the modification of polymeric micellar surface with breast tumor targeting peptide, P18-4.

Microfluidics: a transformational tool for nanomedicine development and production.

Microfluidic devices are mircoscale fluidic circuits used to manipulate liquids at the nanoliter scale. The ability to control the mixing of fluids and the continuous nature of the process make it apt for solvent/antisolvent precipitation of drug-delivery nanoparticles. This review describes the use of numerous microfluidic designs for the formulation and production of lipid nanoparticles, liposomes and polymer nanoparticles to encapsulate and deliver small molecule or genetic payloads. The advantages of microfluidics are illustrated through examples from literature comparing conventional processes such as beaker and T-tube mixing to microfluidic approaches. Particular emphasis is placed on examples of microfluidic nanoparticle formulations that have been tested in vitro and in vivo. Fine control of process parameters afforded by microfluidics, allows unprecedented optimization of nanoparticle quality and encapsulation efficiency. Automation improves the reproducibility and optimization of formulations. Furthermore, the continuous nature of the microfluidic process is inherently scalable, allowing optimization at low volumes, which is advantageous with scarce or costly materials, as well as scale-up through process parallelization. Given these advantages, microfluidics is poised to become the new paradigm for nanomedicine formulation and production.

Polymeric micelles for MCL-1 gene silencing in breast tumors following systemic administration.

AIM: To develop delivery systems for efficient siRNA delivery to breast cancer. METHODS: Poly(ethylene oxide)-block-poly(ϵ-caprolactone-grafted-spermine) (PEO-b-P(CL-g-SP)) micelles were modified with cholesterol group in their core and with RGD4C peptide on their shell. Transfection efficiency of complexed MCL-1 siRNA in MDA-MB-435 was investigated, in vitro and in vivo following intratumoral and intravenous injection. RESULTS: Cholesteryl modification of the core significantly increased the transfection efficiency of PEO-b-P(CL-g-SP)-complexed siRNA, in vitro, but not following intratumoral or intravenous administration, in vivo. Instead, RGD4C modification of the micellar shell enhanced transfection efficiency of complexed MCL-1 siRNA in tumor upon intravenous administration. CONCLUSION: RGD4C-PEO-b-P(CL-g-SP) micelles, without or with cholesterol modification, can provide efficient delivery of siRNA to breast tumors following systemic administration.

Polymeric micelles based on poly(ethylene oxide) and α-carbon substituted poly(ɛ-caprolactone): An in vitro study on the effect of core forming block on polymeric micellar stability, biocompatibility, and immunogenicity.

A series of block copolymers based on methoxy poly(ethylene oxide)-block-poly(ɛ-caprolactone) (PEO-b-PCL), PEO-b-PCL bearing side groups of benzyl carboxylate (PEO-b-PBCL), or free carboxyl (PEO-b-PCCL) on the PCL backbone with increasing degrees of polymerization of the PCL backbone were synthesized. Prepared block copolymers assembled to polymeric micelles by co-solvent evaporation. The physical stability of prepared micelles was assessed by measuring their tendency toward aggregation over time using dynamic light scattering (DLS). The resistance of micelles against dissociation in the presence of a micelle destabilizing agent, i.e., sodium dodecyl sulfate (SDS), was also investigated using DLS. The rate of micellar core degradation was determined using (1)H NMR for polymer molecular weight measurement upon incubation of micelles in PBS (pH=7.4) at 37°C followed by dialysis of the remaining polymer at different time intervals. The effect of pendent group chemistry in the micellar core on the adsorption of serum proteins to micellar structure was then evaluated using Bradford Protein assay kit. Finally, the effect of micellar core structure on the induction of bone marrow derived dendritic cell (BMDC) maturation and secretion of IL-12 was studied as a measure of micellar immunogenicity. The results showed micelle structures from polymers with higher degree of polymerization in the hydrophobic block and/or those with more hydrophobic substituents on the core-forming block, to be more stable. This was reflected by a decreased tendency for micellar aggregation, reduced dissociation of micelles in the presence of SDS, and diminished core degradation. All micelles were shown to have insignificant adsorption of serum protein suggesting that the hydrophilic PEO shell provided sufficient protection of the core. However, the protein adsorption increased with increase in the hydrophobicity and molecular weight of the core-forming block. Irrespective of the micellar core structure, all tested micelles were found to be non-immunogenic in BMDCs.

Engineering of amphiphilic block copolymers for polymeric micellar drug and gene delivery.

The use of nano-delivery systems formed through assembly of synthetic amphiphilic block copolymers (ABCs) in experimental medicine and pharmaceutical sciences is experiencing rapid development. This rapid development is driven by a crucial need in improving the performance of existing therapeutic agents, as well as the necessity for the development of advanced delivery systems for complex new entities such as genes, proteins and other cellular components. The flexibility in the construction of appropriate carriers for the delivery requirements of these complex new "drugs" offered by versatile polymer chemistry provides an undeniable advantage for polymer based nano-delivery systems compared to other colloids in this regard. With seven formulations already in different stages of clinical trials, polymeric micelles are in the front line of drug development among different ABC-based nano-carriers. The success in rapid advancement of polymeric micelles from bench to bedside is owed to the rational engineering of core/shell structure so that the polymeric micellar carrier can meet the requirements for optimum delivery of specific drug(s) in certain disease condition(s). The engineering efforts in this regard have mostly been aimed at providing efficient drug loading, micellar stabilization, and sustained and/or site specific drug release. The objective of this review is to provide an update on different engineering strategies employed to achieve optimum polymeric micellar formulations.