Nano-Assembly of Pamitoyl-Bioconjugated Coenzyme‑A for Combinatorial Chemo-Biologics in Transcriptional Therapy

Pathogenesis, the biological mechanism that leads to the diseased state, of many cancers is driven by interruptions to the role of Myc oncoprotein, a regulator protein that codes for a transcription factor. One of the most significant biological interruptions to Myc protein is noted as its dimerization with Max protein, another important factor of family of transcription factors. Binding of this heterodimer to E-Boxes, enhancer boxes as DNA response element found in some eukaryotes that act as a protein-binding site and have been found to regulate gene expression, are interrupted to regulate cancer pathogenesis. The systemic effectiveness of potent small molecule inhibitors of Myc-Max dimerization has been limited by poor bioavailability, rapid metabolism, and inadequate target site penetration. The potential of gene therapy for targeting Myc can be fully realized by successful synthesis of a smart cargo. We developed a “nuclein” type nanoparticle “siNozyme” (45 ± 5 nm) from nanoassembly of pamitoyl-bioconjugated acetyl coenzyme-A for stable incorporation of chemotherapeutics and biologics to achieve remarkable growth inhibition of human melanoma. Results indicated that targeting transcriptional gene cMyc with siRNA with codelivery of a topoisomerase inhibitor, amonafide caused ∼90% growth inhibition and 95% protein inhibition

Genomic DNA Interactions Mechanize Peptidotoxin-Mediated Anticancer Nanotherapy

Host defense peptides (HDPs) are a class of evolutionarily conserved substances of the innate immune response that have been identified as major players in the defense system in many living organisms. Some of the HDPs are also referred to as peptidotoxins, which offer immense potential for anticancer therapy. However, their therapeutic potential is yet to be fully translated mainly due to their off-target toxicity. Here we show that their nanoenabled delivery may become beneficial in controlling their delivery in intracellular space. We introduced an amphiphilic polymer to synthesize a well-defined, self-assembled, rigid-cored polymeric nanoarchitecture for controlled delivery of three model peptidotoxins, i.e., melittin, TSAP-1, and a negative control peptide of synthetic origin. Interestingly, our results revealed strong interaction of peptidotoxins with duplex plasmid DNA. Extensive biophysical characterization (UV–vis spectroscopy, gel electrophoresis, MTT assay, and flow assisted cell sorting) experimentally verified that peptidotoxins were able to interact with genomic DNA in vitro and in turn influence the cancer cell growth. Thus, we unraveled that, through genomic DNA regulation, peptidotoxins can play a role in cell cycle regulation and exert their anticancer activities

Defined Nanoscale Chemistry Influences Delivery of Peptido-Toxins for Cancer Therapy

<div><p>We present an <i>in-silico-to-in-vitro</i> approach to develop well-defined, self-assembled, rigid-cored polymeric (Polybee) nano-architecture for controlled delivery of a key component of bee venom, melittin. A competitive formulation with lipid-encapsulated (Lipobee) rigid cored micelle is also synthesized. In a series of sequential experiments, we show how nanoscale chemistry influences the delivery of venom toxins for cancer regression and help evade systemic disintegrity and cellular noxiousness. A relatively weaker association of melittin in the case of lipid-based nanoparticles is compared to the polymeric particles revealed by energy minimization and docking studies, which are supported by biophysical studies. For the first time, the authors’ experiment results indicate that melittin can play a significant role in DNA association-dissociation processes, which may be a plausible route for their anticancer activity.</p></div

Release mechanism, systemic toxicity and stability studies.

<p>(a) Complementing activation and (b) melittin leaching behavior of Lipobee and Polybees. Free melittin, LRCM and PRCM were used as controls; (c) optical microscopy images of blood smear untreated (i) and treated with melittin (1:10) (ii), LRCM (1:10) (iii), Lipobee (1:10) (iv), PRCM (1:10) (v) and polybee (1:10) (vi), respectively, (with 20x magnification). Melittin- and Lipobee-treated pig blood in the severely clumped, morphologically distorted state are shown in (ii) and (iv). Insets in (ii) and (iv) show red blood cell morphology to emphasize other similar morphological patterns throughout the sample.</p

Scores of different docking poses of melittin superimposed and energy minimized with lipid or amphiphilic polymer system.

<p>Scores of different docking poses of melittin superimposed and energy minimized with lipid or amphiphilic polymer system.</p

Preparation and physico-chemical characterization studies.

<p>Synthesis and characterization of rigid core micelles and melittin loaded particles: (a) Synthesis of PRCM and Polybee nanoparticles; (b) representative TEM images of Polybee; (c) representative AFM images of Polybee; (d) Synthesis of LRCM and Lipobee nanoparticles; (e) representative TEM images of Lipobee; (c) representative AFM images of Lipobee; (f) UV-vis spectroscopy of melittin, LRCM, PRCM, Lipobee and Polybee; (g) hydrodynamic diameter distribution (number averaged, nm). TEM samples (20 μL) were prepared on formvar-coated carbon grids and negatively stained with uranyl acetate and vacuum dried before performing the microscopy. Samples (20 μL) were drop casted on freshly cleaved mica sheets and air dried for >24h before performing the tapping mode AFM.</p

Hydrodynamic diameter distribution, anhydrous state particle size, particle height and electrophoretic potential distribution of PRCM, Polybee and LRCM and Lipobee in tabular form.

<p>Hydrodynamic diameter distribution, anhydrous state particle size, particle height and electrophoretic potential distribution of PRCM, Polybee and LRCM and Lipobee in tabular form.</p

Functional characterization in vitro.

<p>Representative bright field images of cell growth density and cancer cell morphology variation for MCF-7 (a-c) and MD-MB231 (d-f) after 48 h of incubation treated with melittin, LRCM and Lipobee, (g) IC50 values for various formulations in tabular form; biostatistical analysis on IC50 values for Polybee respect to melittin representing *** for p value < 0.001 and ** for p value < 0.005 after ONE way ANOVA with Bonferroni post test and (h-i) % cell viability variations by different formulation in MD-MB231 and (j-k) MCF-7 cells for (h-j) polymeric and (i-k) lipidic formulations.</p

DNA interaction studies.

<p>(a) Illustration of docked structure of melittin with DNA. (b) Key interactions of melittin with DNA. Melittin is shown as green links. (c) Molcad surface picture of docked structure.</p

Molecular docking studies.

<p>Super-imposition of the five best docking poses of melittin with lecithin PC and PS₆₇-<i>b</i>-PAA₂₇ polymer: (a) Docking poses of melittin to PS₆₇-<i>b</i>-PAA₂₇ polymer; (b) docking poses of melittin to lecithin PC. The best scored pose is in the green linked chains with the following smaller attachments listed in order according to their score as indicated by their color: Magenta, 2nd; yellow, 3rd; white, 4th, and cyan, 5th.</p