Modulating Pharmacokinetics, Tumor Uptake and Biodistribution by Engineered Nanoparticles

Inorganic nanoparticles provide promising tools for biomedical applications including detection, diagnosis and therapy. While surface properties such as charge are expected to play an important role in their in vivo behavior, very little is known how the surface chemistry of nanoparticles influences their pharmacokinetics, tumor uptake, and biodistribution.Using a family of structurally homologous nanoparticles we have investigated how pharmacological properties including tumor uptake and biodistribution are influenced by surface charge using neutral (TEGOH), zwitterionic (Tzwit), negative (TCOOH) and positive (TTMA) nanoparticles. Nanoparticles were injected into mice (normal and athymic) either in the tail vein or into the peritoneum.Neutral and zwitterionic nanoparticles demonstrated longer circulation time via both i.p. and i.v. administration, whereas negatively and positively charged nanoparticles possessed relatively short half-lives. These pharmacological characteristics were reflected on the tumor uptake and biodistribution of the respective nanoparticles, with enhanced tumor uptake by neutral and zwitterionic nanoparticles via passive targeting

Identifying New Therapeutic Targets via Modulation of Protein Corona Formation by Engineered Nanoparticles

We introduce a promising methodology to identify new therapeutic targets in cancer. Proteins bind to nanoparticles to form a protein corona. We modulate this corona by using surface-engineered nanoparticles, and identify protein composition to provide insight into disease development.Using a family of structurally homologous nanoparticles we have investigated the changes in the protein corona around surface-functionalized gold nanoparticles (AuNPs) from normal and malignant ovarian cell lysates. Proteomics analysis using mass spectrometry identified hepatoma-derived growth factor (HDGF) that is found exclusively on positively charged AuNPs ((+)AuNPs) after incubation with the lysates. We confirmed expression of HDGF in various ovarian cancer cells and validated binding selectivity to (+)AuNPs by Western blot analysis. Silencing of HDGF by siRNA resulted s inhibition in proliferation of ovarian cancer cells.We investigated the modulation of protein corona around surface-functionalized gold nanoparticles as a promising approach to identify new therapeutic targets. The potential of our method for identifying therapeutic targets was demonstrated through silencing of HDGF by siRNA, which inhibited proliferation of ovarian cancer cells. This integrated proteomics, bioinformatics, and nanotechnology strategy demonstrates that protein corona identification can be used to discover novel therapeutic targets in cancer

Dynamic Light Scattering (DLS) to determine hydrodynamic diameter (d_H) of AuNPs before and after incubation with OV167 and OSE lysates.

<p>Dynamic Light Scattering (DLS) to determine hydrodynamic diameter (d_H) of AuNPs before and after incubation with OV167 and OSE lysates.</p

Venn diagram demonstrating the difference between the proteins that make up the corona for a) ⁺AuNP, b) ⁻AuNP from the cell lysates and c) the different between proteins in the OV167 and OSE cell lysates.

<p>These differences in the composition of the lysates could be exploited to identify new therapeutic targets in ovarian cancer.</p

Enrichment and functional consequence of HDGF.

<p>a) Western blot confirming the presence of HDGF on ⁺AuNP- corona whereas Hsp90 on ⁻AuNP- corona. Also shown is Hsp70 (known to be present in all NP coronas via MS). b) Expression of HDGF in ovarian cell lines. c) Knockdown of HDGF in A2780 cell line using HDGF-siRNAs (KD-siRNA) and compared with the scrambled control (scRNA); d) Effect of silencing HDGF on proliferation of ovarian cancer cells analyzed by [3H]-thymidine incorporation assay.</p

Selectivity of the proteins bound to positively charged (⁺AuNP) vs negatively charged (⁻AuNP) particles.

<p>Venn diagrams show proteins identified in the protein corona around ⁺AuNP and ⁻AuNP from a) normal OSE cell lysates and b) malignant OV167 cell lysates. The figure clearly depicts the preferential enrichment of low abundance proteins by engineered nanoparticles that were not detectable in the lysates by proteomics analysis. These proteins, which were otherwise undetected, could potentially be new therapeutic targets.</p

Characterization of the AuNP and protein corona made from cell lysates of OSE and OV167.

<p>a) A cartoon showing functionalization of 5 nm gold nanoparticle to create positively charged (⁺AuNP) or negatively charged (⁻AuNP) gold nanoparticles. b) Amount of protein bound on the nanoparticle as determined by Bradford assay. The binding of protein is evident from the increase in surface size via DLS on c) ⁺AuNP and d) ⁻AuNP.</p

Zeta Potential in mV of ⁺AuNP and ⁻AuNP before and after incubation with the lysates of OV167 and OSE, respectively.

<p>Zeta Potential in mV of ⁺AuNP and ⁻AuNP before and after incubation with the lysates of OV167 and OSE, respectively.</p

Understanding Protein–Nanoparticle Interaction: A New Gateway to Disease Therapeutics

Molecular identification of protein molecules surrounding nanoparticles (NPs) may provide useful information that influences NP clearance, biodistribution, and toxicity. Hence, nanoproteomics provides specific information about the environment that NPs interact with and can therefore report on the changes in protein distribution that occurs during tumorigenesis. Therefore, we hypothesized that characterization and identification of protein molecules that interact with 20 nm AuNPs from cancer and noncancer cells may provide mechanistic insights into the biology of tumor growth and metastasis and identify new therapeutic targets in ovarian cancer. Hence, in the present study, we systematically examined the interaction of the protein molecules with 20 nm AuNPs from cancer and noncancerous cell lysates. Time-resolved proteomic profiles of NP-protein complexes demonstrated electrostatic interaction to be the governing factor in the initial time-points which are dominated by further stabilization interaction at longer time-points as determined by ultraviolet–visible spectroscopy (UV–vis), dynamic light scattering (DLS), ζ-potential measurements, transmission electron microscopy (TEM), and tandem mass spectrometry (MS/MS). Reduction in size, charge, and number of bound proteins were observed as the protein-NP complex stabilized over time. Interestingly, proteins related to mRNA processing were overwhelmingly represented on the NP-protein complex at all times. More importantly, comparative proteomic analyses revealed enrichment of a number of cancer-specific proteins on the AuNP surface. Network analyses of these proteins highlighted important hub nodes that could potentially be targeted for maximal therapeutic advantage in the treatment of ovarian cancer. The importance of this methodology and the biological significance of the network proteins were validated by a functional study of three hubs that exhibited variable connectivity, namely, PPA1, SMNDC1, and PI15. Western blot analysis revealed overexpression of these proteins in ovarian cancer cells when compared to normal cells. Silencing of PPA1, SMNDC1, and PI15 by the siRNA approach significantly inhibited proliferation of ovarian cancer cells and the effect correlated with the connectivity pattern obtained from our network analyses

Cystathionine Beta-Synthase (CBS) Contributes to Advanced Ovarian Cancer Progression and Drug Resistance

<div><p>Background</p><p>Epithelial ovarian cancer is the leading cause of gynecologic cancer deaths. Most patients respond initially to platinum-based chemotherapy after surgical debulking, however relapse is very common and ultimately platinum resistance emerges. Understanding the mechanism of tumor growth, metastasis and drug resistant relapse will profoundly impact the therapeutic management of ovarian cancer.</p><p>Methods/Principal Findings</p><p>Using patient tissue microarray (TMA), <i>in vitro</i> and <i>in vivo</i> studies we report a role of of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme in ovarian carcinoma. We report here that the expression of cystathionine-beta-synthase (CBS), a sulfur metabolism enzyme, is common in primary serous ovarian carcinoma. The <i>in vitro</i> effects of CBS silencing can be reversed by exogenous supplementation with the GSH and H₂S producing chemical Na₂S. Silencing CBS in a cisplatin resistant orthotopic model <i>in vivo</i> by nanoliposomal delivery of CBS siRNA inhibits tumor growth, reduces nodule formation and sensitizes ovarian cancer cells to cisplatin. The effects were further corroborated by immunohistochemistry that demonstrates a reduction of H&E, Ki-67 and CD31 positive cells in si-RNA treated as compared to scrambled-RNA treated animals. Furthermore, CBS also regulates bioenergetics of ovarian cancer cells by regulating mitochondrial ROS production, oxygen consumption and ATP generation. This study reports an important role of CBS in promoting ovarian tumor growth and maintaining drug resistant phenotype by controlling cellular redox behavior and regulating mitochondrial bioenergetics.</p><p>Conclusion</p><p>The present investigation highlights CBS as a potential therapeutic target in relapsed and platinum resistant ovarian cancer.</p></div