Jolanta F. Kukowska-Latallo

Nanoparticle Targeting of Anticancer Drug Improves Therapeutic Response in Animal Model of Human Epithelial Cancer

Prior studies suggested that nanoparticle drug delivery might improve the therapeutic response to anticancer drugs and allow the simultaneous monitoring of drug uptake by tumors. We employed modified PAMAM dendritic polymers <5 nm in diameter as carriers. Acetylated dendrimers were conjugated to folic acid as a targeting agent and then coupled to either methotrexate or tritium and either fluorescein or 6-carboxytetramethylrhodamine. These conjugates were injected i.v. into immunodeficient mice bearing human KB tumors that overexpress the folic acid receptor. In contrast to nontargeted polymer, folate-conjugated nanoparticles concentrated in the tumor and liver tissue over 4 days after administration. The tumor tissue localization of the folate-targeted polymer could be attenuated by prior i.v. injection of free folic acid. Confocal microscopy confirmed the internalization of the drug conjugates into the tumor cells. Targeting methotrexate increased its antitumor activity and markedly decreased its toxicity, allowing therapeutic responses not possible with a free drug.

The use of PAMAM dendrimers in the efficient transfer of genetic material into cells

Polyamidoamine (PAMAM) dendrimers have steadily grown in popularity in the past decade in a variety of disciplines, ranging from materials science to biomedicine. This can be attributed in part to their use in applications that range from computer toners to medical diagnostics. PAMAM dendrimers are safe and nonimmunogenic, and can function as highly efficient cationic polymer vectors for delivering genetic material into cells. They have been shown to be as efficient or more efficient than either cationic liposomes or other cationic polymers (e.g. polyethylenimine, polylysine) for in vitro gene transfer. This article will focus on the application of PAMAM dendrimers as a nonviral gene delivery vector from the initial discovery of this capacity to the most recent experimental findings.

The interaction of plasmid DNA with polyamidoamine dendrimers: mechanism of complex formation and analysis of alterations induced in nuclease sensitivity and transcriptional activity of the complexed DNA

The application of synthetic vectors for gene transfer has potential advantages over virus-based systems. However, little is known about the mechanisms involved in binding of synthetic materials to DNA and the nature of the DNA complexes that result from this interaction. Polyamidoamine (PAMAM) dendrimers are unique polymers with defined spherical structure. Dendrimers bind DNA to form complexes that efficiently transfect cells in vitro. We examined the formation of DNA/dendrimer complexes and found it based entirely on charge interaction. Electronmicroscopic examination of the complexes indicated that the majority of the plasmid DNA is contracted into isolated toroids, but also revealed larger, irregular aggregates of polymer and DNA. The binding of plasmid DNA to dendrimer appears to alter the secondary and tertiary structure, but does not fragment the DNA or alter its primary structure. Complexed DNA is protected against degradation by either specific nucleases or cellular extracts containing nuclease activity. While the initiation of transcription in vitro from promoters (for either T7 polymerase or eukaryotic RNA polymerase II) in dendrimer-complexed DNA is inhibited, elongation of the RNA transcript and translation do not appear to be affected. These resemble alterations of the DNA function when complexed with naturally-occurring polycations like non-acetylated histones. However, DNA complexed to dendrimer appears to maintain transcriptional activity while histone complexes at similar charge ratios do not. These results elucidate some aspects of the interaction between PAMAM dendritic polymers and DNA, and could lead to improvements in the design of polymers or formation of DNA complexes that will increase the efficiency of non-viral gene transfer.

Targeted gadolinium-loaded dendrimer nanoparticles for tumor-specific magnetic resonance contrast enhancement

A target-specific MRI contrast agent for tumor cells expressing high affinity folate receptor was synthesized using generation five (G5) of polyamidoamine (PAMAM) dendrimer. Surface modified dendrimer was functionalized for targeting with folic acid (FA) and the remaining terminal primary amines of the dendrimer were conjugated with the bifunctional NCS-DOTA chelator that forms stable complexes with gadolinium (Gd III). Dendrimer-DOTA conjugates were then complexed with GdCl3 followed by ICP-OES as well as MRI measurement of their longitudinal relaxivity (T1 s−1 mM−1) of water. In xenograft tumors established in immunodeficient (SCID) mice with KB human epithelial cancer cells expressing folate receptor (FAR), the 3D MRI results showed specific and statistically significant signal enhancement in tumors generated with targeted Gd(III)-DOTA-G5-FA compared with signal generated by non-targeted Gd(III)-DOTA-G5 contrast nanoparticle. The targeted dendrimer contrast nanoparticles infiltrated tumor and were retained in tumor cells up to 48 hours post-injection of targeted contrast nanoparticle. The presence of folic acid on the dendrimer resulted in specific delivery of the nanoparticle to tissues and xenograft tumor cells expressing folate receptor in vivo. We present the specificity of the dendrimer nanoparticles for targeted cancer imaging with the prolonged clearance time compared with the current clinically approved gadodiamide (Omniscan™) contrast agent. Potential application of this approach may include determination of the folate receptor status of tumors and monitoring of drug therapy.

Intravascular and Endobronchial DNA Delivery to Murine Lung Tissue Using a Novel, Nonviral Vector

Gene transfer to the lung can be achieved via either the airway or the pulmonary vasculature. We evaluated gene transfer and expression by intravascular and endobronchial routes, using DNA complexed with G9 PAMAM dendrimer or naked plasmid DNA. Intravascular tail vein delivery of dendrimer-complexed pCF1CAT plasmid resulted in high levels of transgene expression in the lung at 12 and 24 hr, followed by a second peak of expression 3 to 5 days after administration. After intravenous administration of the complexes, CAT expression was never observed in organs other than the lung. There were only minimal levels of CAT protein expressed in the lung after intravenous administration of naked plasmid DNA. Repeated intravascular doses of the dendrimer-complexed plasmid, administered four times at 4-day intervals, maintained expression at 15-25% of peak concentrations achieved after the initial dose. Endobronchial delivery of naked pCF1CAT plasmid produced significant amounts of CAT protein in the lung. Comparison of intratracheal and intranasal routes resulted in similar expression levels of CAT in the lung and trachea. However, in juxtaposition to vascular delivery, intranasal delivery of dendrimer-complexed plasmid DNA gave lower levels of CAT expression than that observed with naked plasmid DNA. In situ localization of CAT enzymatic activity suggested that vascular administration seemed to achieve expression in the lung parenchyma, mainly within the alveoli, while endobronchial administration primarily targeted bronchial epithelium. Our results show that intravenously administered G9 dendrimer is an effective vector for pulmonary gene transfer and that transgene expression can be prolonged by repeated administration of dendrimer-complexed DNA.

Development of immune response that protects mice from viral pneumonitis after a single intranasal immunization with influenza A virus and nanoemulsion.

Nanoemulsion, a water-in-oil formulation stabilized by small amounts of surfactant, is non-toxic to mucous membranes and produces biocidal activity against enveloped viruses. We evaluated nanoemulsion as an adjuvant for mucosal influenza vaccines. Mice (C3H/HeNHsd strain) were vaccinated intranasally with 5 x 10(5) plaque forming units (pfu) of influenza A virus (Ann Arbor/6/60 strain) and a nanoemulsion mixture. The mice were challenged on day 21 after immunization with an intranasal lethal dose of 2 x 10(5) pfu of virus. Animals vaccinated with the influenza A/nanoemulsion mixture were completely protected against infection, while animals vaccinated with either formaldehyde-killed virus or nanoemulsion alone developed viral pneumonitis and died by day 6 after the challenge. Mice vaccinated with virus/nanoemulsion mixture had rapid cytokine responses followed by high levels of specific anti-influenza immunoglobulin G (IgG) and immunoglobulin A (IgA) antibodies. Specificity of the immune response was confirmed by assessment of the proliferation and cytokine production in splenocytes. This paper demonstrates that nanoemulsion can be employed as a non-toxic mucosal adjuvant for influenza virus vaccine.

Preclinical antitumor efficacy evaluation of dendrimer-based methotrexate conjugates

Our previous studies have demonstrated the in-vitro and in-vivo targeting of a generation-5 (G5) dendrimer-based multifunctional conjugate, which used folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. For the synthesized G5-FA-MTX nanodevice conjugate to be clinically applicable as a cancer therapeutic drug, it is important that the compound elicits cytotoxicity specifically and consistently. The aim of this work was to evaluate four independently synthesized batches of G5-FA-MTX conjugates for their cytotoxic potential and specificity. For determination of specificity, we have used a unique ‘coculture’ assay in which FA receptor-positive and FA receptor-negative cells were cultured together and have examined the preferential killing of the former. The results of our study show the batch-to-batch consistency and specificity of the G5-FA-MTX nanodevice in the preferential killing of FA receptor-positive cells. The coculture assay shows the consistency of the four different G5-FA-MTX conjugate lots in the specific killing of targeted cells. Further in-vivo studies are, however, necessary to prove the clinical potential of this targeted therapeutic nanodevice.

Targeting the efficacy of a dendrimer-based nanotherapeutic in heterogeneous xenograft tumors in vivo.

Our earlier studies have shown the in vitro and in vivo targeting of a generation 5 (G5) dendrimer-based multifunctional conjugate that contained folic acid (FA) as the targeting agent and methotrexate (MTX) as the chemotherapeutic drug. To clinically apply the synthesized G5-FA-MTX nanotherapeutic, it is important that the anticancer conjugate elicits cytotoxicity specifically and consistently. Toward this objective, we evaluated the large-scale synthesis of a G5-FA-MTX conjugate (Lot # 123–34) for its cytotoxic potential and specificity in vitro and in vivo. The cytotoxicity and specificity were tested by using a coculture assay in which FA receptor-expressing and nonexpressing cells (KB and SK-BR-3 cells, respectively) were cultured together and preferential killing was examined. The in-vitro data were compared with the in-vivo data obtained from a heterogeneous xenograft tumor model. The animal model of the artificial heterogeneous xenograft tumor showed that the nanotherapeutic was preferentially cytotoxic to KB cells.

The facile synthesis of multifunctional PAMAM dendrimer conjugates through copper-free click chemistry.

The facile conjugation of three azido modified functionalities, namely a therapeutic drug (methotrexate), a targeting moiety (folic acid), and an imaging agent (fluorescein) with a G5 PAMAM dendrimer scaffold with cyclooctyne molecules at the surface through copper-free click chemistry is reported. Mono-, di-, and tri-functional PAMAM dendrimer conjugates can be obtained via combinatorial mixing of different azido modified functionalities simultaneously or sequentially with the dendrimer platform. Preliminary flow cytometry results indicate that the folic acid targeted nanoparticles are efficiently binding with KB cells.

Combination of Electroporation and DNA/Dendrimer Complexes Enhances Gene Transfer into Murine Cardiac Transplants

Electroporation is a new gene delivery method to increase gene transfer and expression in vivo. Starburst polyamidoamine dendrimers have been demonstrated to augment gene expression in vitro and in vivo. We hypothesized that the combination of electroporation and dendrimer could enhance the gene transfer and gene expression in cardiac transplants. After immersion in DNA/dendrimer complexes or intracoronary transfer of DNA/dendrimer complexes, both nonvascularized and vascularized syngeneic cardiac grafts, respectively, were subjected to serial electrical pulses before transplantation. β‐Galactosidase reporter gene expression in the graft was determined by X‐Gal staining. Gene expression was enhanced 10‐ to 45‐fold in grafts immersed in DNA/dendrimer complexes, or after intracoronary transfer of DNA/dendrimer complexes, and subjected to 20 square wave 25‐ms pulses with a strength of 200 V/cm. The combination of electroporation and DNA/dendrimer complexes may provide a novel approach to enhance gene transfer and gene expression ex vivo.