Zoltan Szekely

Pharmaceutical and Toxicological Properties of Engineered Nanomaterials for Drug Delivery

Novel engineered nanomaterials (ENMs) are being developed to enhance therapy. The physicochemical properties of ENMs can be manipulated to control/direct biodistribution and target delivery, but these alterations also have implications for toxicity. It is well known that size plays a significant role in determining ENM effects since simply nanosizing a safe bulk material can render it toxic. However, charge, shape, rigidity, and surface modifications also have a significant influence on the biodistribution and toxicity of nanoscale drug delivery systems (NDDSs). In this review, NDDSs are considered in terms of platform technologies, materials, and physical properties that impart their pharmaceutical and toxicological effects. Moving forward, the development of safe and effective nanomedicines requires standardized protocols for determining the physical characteristics of ENMs as well as assessing their potential long-term toxicity. When such protocols are established, the remarkable promise of nanomedicine to improve the diagnosis and treatment of human disease can be fulfilled.

Drug delivery strategies and systems for HIV/AIDS pre-exposure prophylaxis and treatment.

The year 2016 will mark an important milestone - the 35th anniversary of the first reported cases of HIV/AIDS. Antiretroviral Therapy (ART) including Highly Active Antiretroviral Therapy (HAART) drug regimens is widely considered to be one of the greatest achievements in therapeutic drug research having transformed HIV infection into a chronically managed disease. Unfortunately, the lack of widespread preventive measures and the inability to eradicate HIV from infected cells highlight the significant challenges remaining today. Moving forward there are at least three high priority goals for anti-HIV drug delivery (DD) research: (1) to prevent new HIV infections from occurring, (2) to facilitate a functional cure, i.e., when HIV is present but the body controls it without drugs and (3) to eradicate established infection. Pre-exposure Prophylaxis (PrEP) represents a significant step forward in preventing the establishment of chronic HIV infection. However, the ultimate success of PrEP will depend on achieving sustained antiretroviral (ARV) tissue concentrations and will require strict patient adherence to the regimen. While first generation long acting/extended release (LA/ER) DD Systems (DDS) currently in development show considerable promise, significant DD treatment and prevention challenges persist. First, there is a critical need to improve cell specificity through targeting in order to selectively achieve efficacious drug concentrations in HIV reservoir sites to control/eradicate HIV as well as mitigate systemic side effects. In addition, approaches for reducing cellular efflux and metabolism of ARV drugs to prolong effective concentrations in target cells need to be developed. Finally, given the current understanding of HIV pathogenesis, next generation anti-HIV DDS need to address selective DD to the gut mucosa and lymph nodes. The current review focuses on the DDS technologies, critical challenges, opportunities, strategies, and approaches by which novel delivery systems will help iterate towards prevention, functional cure and eventually the eradication of HIV infection.

Optimal structural design of mannosylated nanocarriers for macrophage targeting.

Macrophages are involved in a number of diseases, such as HIV infection/AIDS, tuberculosis, tumor development and atherosclerosis. Macrophages possess several cell surface receptors (e.g., the mannose receptor, MR) that may serve as drug delivery cellular portals for nanocarriers (NCs). In this study, the optimal structural configuration for cell uptake of mannosylated poly(ethylene glycol)-conjugate type NCs was determined. A series of NCs were synthesized to systematically evaluate the effects of the number of mannose units (Man), the PEG carrier size and the mPEG spacer length between adjacent mannose units on NC uptake into MR-expressing J774.E murine macrophage-like cells. Among NCs with 0, 1, 2 or 4 units of mannose, the uptake of (Man)2-NC was the highest, suggesting a trade-off between avidity and NC-MR clustering on the cell surface that sterically hinders endocytosis. This optimal (Man)2-NC configuration was built into subsequent NCs to optimize the other two parameters, PEG carrier size and spacer length. NCs with 0, 5, 12, 20, 30 or 40 kDa linear PEG carriers showed an inverse relationship between PEG size and uptake. The 12 kDa PEG carrier was chosen for investigating the third parameter, the Man-Man distance, since it may represent the best trade off (i.e., tissue penetration vs. systemic clearance) for in vivo macrophage targeting. Three (Man)2-PEG12kDa NCs with different Man-Man distances (39, 56 or 89Å) were synthesized. The uptake of the NC with the 56Å distance between mannoses was four- and two-fold higher than NCs with 39Å and 89Å distances, respectively. Confocal microscopy confirmed that the optimized (Man)2-PEG12kDa NC with the 56Å Man-Man distance was internalized via endocytosis consistent with temperature-dependent active uptake. In conclusion, the optimal NC structural parameters for targeting the MR on macrophage-like J774.E cells are (i) a small PEG polymer carrier, (ii) two mannose units per NC and (iii) a 56Å distance between adjacent mannose units.

Responsive foams for nanoparticle delivery

We have developed responsive foam systems for nanoparticle delivery. The foams are easy to make, stable at room temperature, and can be engineered to break in response to temperature or moisture. Temperature-responsive foams are based on the phase transition of long chain alcohols and could be produced using medical grade nitrous oxide as a propellant. These temperature-sensitive foams could be used for polyacrylic acid (PAA)-based nanoparticle delivery. We also discuss moisture-responsive foams made with soap pump dispensers. Polyethylene glycol (PEG)-based nanoparticles or PMMA latex nanoparticles were loaded into Tween 20 foams and the particle size was not affected by the foam formulation or foam break. Using biocompatible detergents, we anticipate this will be a versatile and simple approach to producing foams for nanoparticle delivery with many potential pharmaceutical and personal care applications.

Activated matriptase as a target to treat breast cancer with a drug conjugate

The antitumor effects of a novel antibody drug conjugate (ADC) was tested against human solid tumor cell lines and against human triple negative breast cancer (TNBC) xenografts in immunosuppressed mice. The ADC targeting activated matriptase of tumor cells was synthesized by using the potent anti-tubulin toxin, monomethyl auristatin-E linked to the activated matriptase-specific monoclonal antibody (M69) via a lysosomal protease-cleavable dipeptide linker. This ADC was found to be cytotoxic against multiple activated matriptase-positive epithelial carcinoma cell lines in vitro and markedly inhibited growth of triple negative breast cancer xenografts and a primary human TNBC (PDX) in vivo. Overexpression of activated matriptase may be a biomarker for response to this ADC. The ADC had potent anti-tumor activity, while the unconjugated M69 antibody was ineffective in a mouse model study using MDA-MB-231 xenografts in mice. Treatment of a human TNBC (MDA-MB-231) showed potent anti-tumor effects in combination with cisplatin in mice. This ADC alone or in combination with cisplatin has the potential to improve the treatment outcomes of patients with TNBC as well as other tumors overexpressing activated matriptase.

The effect of size and polymer architecture of doxorubicin–poly(ethylene) glycol conjugate nanocarriers on breast duct retention, potency and toxicity

&NA; Although systemic administration of chemotherapeutic agents is routinely used for treating invasive breast cancer, the only therapeutic options for ductal carcinoma in situ (DCIS) are surgery and radiation. Treating DCIS by delivering drugs locally to the affected milk duct offers significant advantages over systemic administration, including reduced systemic and breast toxicities, as well as a greatly reduced need for surgery and radiation. In this study, mammary gland retention and toxicity of intraductally administered poly(ethylene) glycol‐doxorubicin (PEG‐DOX) polymeric conjugate nanocarriers of varying molecular sizes and architectures were investigated. Nanocarriers were formed by conjugating one or more copies of doxorubicin to PEG polymers, of varying molecular weights (5, 10, 20, and 40 kDa) and architectures (linear, four‐arm and eight‐arm). Cytotoxicity against MCF7 cells, a human breast cancer cell line, was assessed, and IC50 values were calculated. The nanocarriers were intraductally administered into the mammary glands of female retired breeder Sprague‐Dawley rats. Whole body images were captured using in vivo optical imaging, and changes in ductal structure as well local inflammation were monitored. Fluorescence intensities were monitored, over time, to evaluate nanocarrier mammary gland retention half‐lives (t1/2). The IC50 values of PEG‐DOX nanocarriers against MCF7 cells were 40 kDa PEG‐(DOX)4 (1.23 &mgr;M) < 5 kDa PEG‐DOX (1.76 &mgr;M) < 40 kDa PEG‐(DOX)8 (3.49 &mgr;M) < 10 kDa PEG‐DOX (3.86 &mgr;M) < 20 kDa PEG‐DOX (8.96 &mgr;M) < 40 kDa PEG‐DOX (18.11 &mgr;M), whereas the IC50 of free DOX was only 0.14 &mgr;M. The t1/2 of linear 5, 20, and 40 kDa nanocarriers were 2.2 ± 0.3, 3.6 ± 0.6, and 13.1 ± 3.4 h, whereas the retention t1/2 of 4‐ and 8‐arm 40 kDa nanocarriers were 14.9 ± 5.6 h and 11.9 ± 2.9 h, respectively. The retention t1/2 of free doxorubicin was 2.0 ± 0.4 h, which was significantly shorter than that of the linear and branched 40 kDa PEG‐DOX nanocarriers. Increased molecular weight and decreased branching both demonstrated a strong correlation to enhanced mammary gland retention. Intraductally administered free doxorubicin resulted in ductal damage, severe inflammation and generation of atypical cell neoplasms, whereas PEG‐DOX nanocarriers induced only minor and transient inflammation (i.e., damaged epithelial cells and detached cellular debris). The 40 kDa 4‐arm PEG‐DOX nanocarrier demonstrated the longest ductal retention half‐life, the lowest IC50 (i.e., most potent), and minimal ductal damage and inflammation. The current results suggest that PEG‐DOX nanocarriers with prolonged ductal retention may present the best option for intraductal treatment of DCIS, due to their low local toxicity and potential for sustained therapeutic effect. Graphical abstract Figure. No caption available.

Modeling and antitumor studies of a modified L–penetratin peptide targeting E2F in lung cancer and prostate cancer

E2F1-3a overexpression due to amplification or to mutation or loss of the retinoblastoma gene, induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more of these activating E2Fs is a recognized target in cancer therapeutics. In previous studies we identified by phage display, a novel 7-mer peptide (PEP) that bound tightly to an immobilized consensus E2F1 promoter sequence, and when conjugated to penetratin to increase its uptake into cells, was cytotoxic to several malignant cell lines and human prostate and small cell lung cancer xenografts. Based on molecular simulation studies that showed that the D-Arg penetratin peptide (D-Arg PEP) secondary structure is more stable than the L-Arg PEP, the L-Arg in the peptide was substituted with D-Arg. In vitro studies confirmed that it was more stable than the L- form and was more cytotoxic as compared to the L-Arg PEP when tested against the human castrate resistant cell line, DU145 and the human lung cancer H196 cell line. When encapsulated in PEGylated liposomes, the D-Arg-PEP potently inhibited growth of the DU145 xenograft in mice. Our findings validate D- Arg PEP, an inhibitor of E2F1and 3a transcription, as an improved second generation drug candidate for targeted molecular therapy of cancers with elevated levels of activated E2F(s).

Evaluation of intraductal delivery of poly(ethylene glycol)-doxorubicin conjugate nanocarriers for the treatment of ductal carcinoma in situ (DCIS)-like lesions in rats: PEG-DOX NANOCARRIERS FOR TREATING DCIS

Ductal carcinoma in situ is the most commonly diagnosed early stage breast cancer. The efficacy of intraductally delivered poly(ethylene glycol)‐doxorubicin (PEG‐DOX) nanocarriers, composed of one or more DOX conjugated to various PEG polymers, was investigated in an orthotopic ductal carcinoma in situ‐like rat model. In vitro cytotoxicity was evaluated against 13762 Mat B III cells using MTT assay. The orthotopic model was developed by inoculating cancer cells into mammary ducts of female Fischer 344 retired breeder rats. The ductal retention and in vivo antitumour efficacy of two of the six nanocarriers (5 kDa PEG‐DOX and 40 kDa PEG‐(DOX)4) were investigated based on in vitro results. Mammary retention of DOX and PEG‐DOX nanocarriers was quantified using in vivo imaging. Histopathologic effects of DOX and PEG‐DOX nanocarriers on mammary ductal structure were also investigated. Cytotoxicities of small linear PEG‐DOX nanocarriers (5 and 10 kDa) were not different from DOX whereas larger PEG‐DOX nanocarriers showed reduced potency. The order of mammary retention was 40 kDa PEG‐(DOX)4 > 5 kDa PEG‐DOX >> DOX, in normal and tumour‐bearing rats. Intraductally administered PEG‐DOX nanocarriers and DOX were effective in reducing tumour incidence and increasing survival rate, with no significant differences found among the three treatment groups. However, nanocarriers administered intravenously at the same doses were not effective, and intraductally administered free DOX caused severe local toxicity. Intraductal administration of PEG‐DOX nanocarriers is effective and less toxic than that of free DOX, as well as IV DOX/PEG‐DOX. Furthermore, PEG‐DOX nanocarriers demonstrate the added benefit of prolonging DOX ductal retention, which would necessitate less frequent dosing.

Design and evaluation of a CXCR4 targeting peptide 4DV3 as an HIV entry inhibitor and a ligand for targeted drug delivery

ABSTRACT The feasibility of utilizing the cell surface chemokine receptor CXCR4 for human immunodeficiency virus (HIV) entry inhibition and as an intracellular portal for targeted drug delivery was evaluated. Novel DV3 ligands (1DV3, 2DV3, and 4DV3) were designed, synthesized and conjugated to various probes (fluorescein isothiocyanate (FITC) or biotin) and cargos with sizes ranging from 10 to 50nm (polyethylene glycol (PEG), streptavidin, and a polymeric nanoparticle). 4DV3 conjugated probes inhibited HIV‐1 entry into the CXCR4‐expressing reporter cell line TZM‐bl (IC50 at 553nM) whereas 1DV3 and 2DV3 did not. 4DV3 also inhibited binding of anti‐CXCR4 antibody 44,708 to TZM‐bl cells with nanomolar potency, while the small‐molecule CXCR4 antagonist AMD3100 did not. Molecular modeling suggested simultaneous binding of a single 4DV3 molecule to four CXCR4 molecules. Differences in CXCR4‐binding sites could explain the discrete inhibitory effects observed for 4DV3, the 44,708 antibody and AMD3100. In the Sup‐T1 cell chemotaxis assay, the 4DV3 ligand functioned as a CXCR4 allosteric enhancer. In addition, 4DV3 ligand‐conjugated cargos with sizes ranging from 10 to 50nm were taken up into CXCR4‐expressing Sup‐T1 and TZM‐bl cells, demonstrating that CXCR4 could serve as a drug delivery portal for nanocarriers. The uptake of 4DV3 functionalized nanocarriers combined with the allosteric interaction with CXCR4 suggests enhanced endocytosis occurs when 4DV3 is the targeting ligand. The current results indicate that 4DV3 might serve as a prototype for a new type of dual function ligand, one that acts as a HIV‐1 entry inhibitor and a CXCR4 drug delivery targeting ligand.

Abstract 1237: Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer

BACKGROUND: Mutation or inactivation of the retinoblastoma protein is frequently involved in prostate cancer tumorigenesis resulting in overexpression/deregulation of E2F activity. E2F1-3a overexpression induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more activating E2Fs is a recognized target in cancer therapeutics. In our previous studies we showed that a novel penetratin conjugated 7-mer peptide (PEP) bound tightly to an immobilized consensus E2F1 promoter sequence, was cytotoxic at low micro molar concentrations to many malignant cell lines and as the PEP was unstable in serum, the PEP was encapsulated in PEGylated liposomes and treatment of tumor xenografts of small cell lung cancer H-69 and DU145 tumors propagated in mice caused tumor regression. OBJECTIVE: To determine the antitumor activity and stability of two different modified penetratin peptides: D-Arg PEP (substituting L-Arginine with D-Arginine in the peptide sequence) and N-acetylated as well as C-methylated PEP analog. METHODS: DU145 (prostate cancer) and H196 (small cell lung cancer) cells were used. To compare the efficacy of the peptides, we tested the IC50s of peptides at different time points using the MTS assay. Drug combination experiment results were analyzed using the combination index (CI) method. Peptide conformational studies were carried out using the Amber 12 suite of biomolecular simulation programs. RESULTS: Molecular simulation studies showed that the D-Arg PEP secondary structure is more stable than the L-Arg peptide structure in water. D-Arg PEP was more potent compared to L-Arg PEP, and it was also found to be more resistant to degradation by serum proteases than the L-form. The other modified form, N-acetylated, C-methylated PEP was marginally more effective than the unmodified PEP. Drug combination studies showed that the D-Arg PEP in combination with docetaxel, caused synergistic cytotoxicity against DU 145 cells. Our findings validate D-Arg peptide, an inhibitor of E2F1and 3a transcription, as a drug candidate for targeted molecular therapy of prostate cancers with elevated levels of activated E2F’s. Studies in progress are evaluating the combination of the PEGylated liposome encapsulated D-Arg PEP in combination with docetaxel against DU145 xenografts and against primary prostate cancer cells. Supported in part by a grant from the Lung Cancer Research Foundation. Citation Format: Tazeem Shaik, Nitu Bansal, Nadine Johnson Farley, John Kerrigan, Olga Garbuzenko, Tamara Minko, Emine Abali, Zoltan Szekely, Kathleen Scotto, Debabrata Banerjee, Joseph Bertino. Antitumor studies of an E2f1 promoter sequence binding peptide - penetratin conjugate as a molecule targeting E2f in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1237. doi:10.1158/1538-7445.AM2015-1237