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Polyelectrolyte complexes (PECs) are self-assembling nano-sized constructs that offer several advantages over traditional nanoparticle carriers including controllable size, biodegradability, biocompatibility, and lack of toxicity, making them particularly appealing as tools for drug delivery. Here, we discuss potential application of PECs for drug delivery to the slightly acidic tumor microenvironment, a pH in the range of 6.5–7.0. Poly(l-glutamic acid) (En), poly(l-lysine) (Kn), and a copolymer composed of histidine-glutamic acid repeats ((HE)n) were studied for their ability to form PECs, which were analyzed for size, polydispersity, and pH sensitivity. PECs showed concentration dependent size variation at residue lengths of E51/K55 and E135/K127, however, no complexes were observed when E22 or K21 were used, even in combination with the longer chains. (HE)20/K55 PECs could encapsulate daunomycin, were stable from pH 7.4–6.5, and dissociated completely between pH 6.5–6.0. Conversely, the E51-dauno/K55 PEC dissociated between pH 4.0 and 3.0. These values for pH-dependent particle dissociation are consistent with the pKa’s of the ionizable groups in each formulation and indicate that the specific pH-sensitivity of (HE)20-dauno/K55 PECs is mediated by incorporation of histidine. This response within a pH range that is physiologically relevant to the acidic tumors suggests a potential application of these PECs in pH-dependent drug delivery.

The injury-triggered reocclusion (restenosis) of arteries treated with angioplasty to relieve atherosclerotic obstruction remains a challenge due to limitations of existing therapies. A combination of magnetic guidance and affinity-mediated arterial binding can pave the way to a new approach for treating restenosis by enabling efficient site-specific localization of therapeutic agents formulated in magnetizable nanoparticles (MNPs) and by maintaining their presence at the site of arterial injury throughout the vulnerability period of the disease. In these studies, we investigated a dual-targeted antirestenotic strategy using drug-loaded biodegradable MNPs, surface-modified with a fibrin-avid peptide to provide affinity for the injured arterial wall. The MNPs were characterized with regard to their magnetic properties, efficiency of surface functionalization, disassembly kinetics, and interaction with fibrin-coated substrates. The antiproliferative effects of MNPs formulated with paclitaxel were studied in vitro using a fetal cell line (A10) exhibiting the defining characteristics of neointimal smooth muscle cells. Animal studies examined the efficiency of combined (physical/affinity) MNP targeting to stented arteries in Sprague Dawley rats using fluorimetric analysis and fluorescent in vivo imaging. The antirestenotic effect of the dual-targeted therapy was determined in a rat model of in-stent restenosis 28 days post-treatment. The results showed that MNPs can be efficiently functionalized to exhibit a strong binding affinity using a simple two-step chemical process, without adversely affecting their size distribution, magnetic properties, or antiproliferative potency. Dual-targeted delivery strongly enhanced the localization and retention of MNPs in stented carotid arteries up to 7 days post-treatment, while minimizing redistribution of the carrier particles to peripheral tissues. Of the two targeting elements, the effect of magnetic guidance was shown to dominate arterial localization (p = 0.004 vs. 0.084 for magnetic targeting and peptide modification, respectively), consistent with the magnetically driven MNP accumulation step defining the extent of the ultimate affinity-mediated arterial binding and subsequent retention of the carrier particles. The enhanced arterial uptake and sustained presence of paclitaxel-loaded MNPs at the site of stent deployment were associated with a strong inhibition of restenosis in the rat carotid stenting model, with both the neointima-to-media ratio (N/M) and % stenosis markedly reduced in the dual-targeted treatment group (1.62 ± 0.2 and 21 ± 3 vs. 2.17 ± 0.40 and 29 ± 6 in the control animals; p < 0.05). We conclude that the dual-targeted delivery of antirestenotic agents formulated in fibrin-avid MNPs can provide a new platform for the safe and effective treatment of in-stent restenosis.

ABSTRACT Purpose Cells modified with magnetically responsive nanoparticles (MNP) can provide the basis for novel targeted therapeutic strategies. However, improvements are required in the MNP design and cell treatment protocols to provide adequate magnetic properties in balance with acceptable cell viability and function. This study focused on select variables controlling the uptake and cell compatibility of biodegradable polymer-based MNP in cultured endothelial cells. Methods Fluorescent-labeled MNP were formed using magnetite and polylactide as structural components. Their magnetically driven sedimentation and uptake were studied fluorimetrically relative to cell viability in comparison to non-magnetic control conditions. The utility of surface-activated MNP forming affinity complexes with replication-deficient adenovirus (Ad) for transduction achieved concomitantly with magnetic cell loading was examined using the green fluorescent protein reporter. Results A high-gradient magnetic field was essential for sedimentation and cell binding of albumin-stabilized MNP, the latter being rate-limiting in the MNP loading process. Cell loading up to 160 pg iron oxide per cell was achievable with cell viability >90%. Magnetically driven uptake of MNP-Ad complexes can provide high levels of transgene expression potentially useful for a combined cell/gene therapy. Conclusions Magnetically responsive endothelial cells for targeted delivery applications can be obtained rapidly and efficiently using composite biodegradable MNP.

This dissertation outlines work completed on formulating and characterizing protein and peptide nanoparticles, with the ultimate goal of targeting these nanoparticles for pH-dependent drug delivery in the acidic tumor microenvironment. This study begins in Chapter Two with a basic exploration of the complexities and limitations of polyelectrolyte complex formation between poly(Glutamic-Acid) and poly(Lysine), as anion and cation, respectively. These complexes are shown to be highly controllable in size and dispersity, and a critical size limitation of 20 amino-acids is reported for successful PEC formation. This concept is then carried into Chapter Three, where the pH-responsive copolymer HEn is paired with poly(Lysine) to form a peptide nanoparticle capable of encapsulating the drug daunomycin, and degrading specifically in response to a decrease in pH to between 6.0-6.5. The next chapters take a slightly different approach, and focus on obtaining a change in nanoparticle surface charge in response to a decreased pH. Chapter Four discusses a novel formulation method for heat-stabilized albumin-only nanoparticles, which removes any need for use of toxic cross-linkers, and provides numerous opportunities for surface modification chemistries. These heat-stabilized nanoparticles are then surface-modified in Chapter Five with various analogues of imidazole, all of which have a pKa of approximately 6.5, allowing them to serve as biologic pH-switches. These surface modifications all result in similar in vitro cell association. To further analyze the potential for imidazole as a physiologically relevant pH-switch, Chapter Six looks at a direct comparison between heat-stabilized nanoparticles that were unmodified, particles modified with the single-imidazole DAH moiety, and particles modified with the di-imidazole DIPA moiety. The DIPA-modified nanoparticles both quantitatively and qualitatively demonstrate increased pH-sensitivity at pH 6.5 compared to 7.4, and increase in vitro cell association compared to the DAH or unmodified nanoparticles, making the DIPA-modified nanoparticles of particular interest for further study in targeting the acidic tumor microenvironment.