Tumor-penetrating acetalated dextran nanoparticles capable of tandem delivery of agents for the treatment of lung cancer

The overall survival rate for patients with lung cancer is still low and many affected patients are not eligible for the first-line treatments (surgery, chemotherapy, and radiation) for non-small cell lung cancer (NSCLC) due to severe side effects. Paclitaxel (PTX) and cisplatin (CDDP) are two of the most commonly utilized drugs in the treatment of NSCLC. These drugs will be encapsulated in tumor-penetrating polymeric nanoparticles (NP) for application in the treatment of lung cancer. Due to the limitations in the NP system itself where particles are often unable to penetrate into the tumor parenchyma to deliver its dose of drug, little has been seen in terms of an increase in clinical outcome for cancer patients treated with nanoparticles although the significant effort that has gone into the study of chemotherapeutic-loaded nanoparticles. Therefore, there is an imperative need for the development of system capable of tumor penetration. Our aim is to develop and optimize peptide-conjugated polymer nanoparticles which can deliver tandem anticancer agents capable of enhancing the targeting and treatment of NSCLC. Acetalated dextran (Ac-Dex) will be used to encapsulate both PTX and CDDP in NPs which in turn will be conjugated with the tumor-penetrating peptide iRGD. This multifunctional particle will not only release PTX and CDDP in tandem, but will be capable of tumor penetration through a mechanism imparted by the peptide. The parameters of the emulsion-based NP system (size, shape, drug loading, peptide-conjugation, cytotoxicity, penetration into NSCLC tumor spheroids, etc.) have been optimized to ensure effective targeting and delivery.

However, the successful delivery of paclitaxel faces many challenges due to its lipophilic nature and high protein affinity, which often results in low bioavailability.
Solubilization of drugs such as PTX, enhanced tumor accumulation, and toxicity attenuation of such therapeutics can be enhanced via targeted delivery to tumors using nanoparticles (NPs). For example, chemotherapeutic drugs such as PTX and doxorubicin have been encapsulated singularly in micelles (Jinhyang 2012) and nanoliposomes (Zhang 2011), thereby increasing the efficacy of radiation therapy against in vitro lung cancer cells and in animals, demonstrating the success of nanoparticle-mediated treatment of cancer. Current cancer treatment, however, revolves around the tandem delivery of PTX and therapeutics such as imatinib, doxorubicin, and carboplatin. Unfortunately, multi-drug delivery is not easily achieved with single-agent delivery systems demonstrating the need for a nanoparticle system capable of delivering multiple drugs in tandem directly to the tumor site(s).
While the enhanced permeability and retention (EPR) effect has served as a key rationale for using nanoparticles to treat solid tumors, as it allows for increased uptake of NPs at the tumor site, it often does not enable uniform delivery of these particles to all regions of tumors. Tumor-penetrating peptides show great promise in overcoming the relevant physiological barriers preventing accumulation and penetrating into the avascular region of tumors. iRGD (internalizing RGD, CRGDKDPDC), has been shown to effectively target and penetrate both in vitro multicellular spheroids (MCS) and in vivo animal model tumors when conjugated to drugs or nanoparticles (Sugahara 2010). For the described research, I hypothesize that iRGD-conjugated nanoparticles containing PTX and imatinib will more effectively home to and penetrate lung cancer tumor spheroids for more effective treatment of this dismal disease.
In particular, acetalated dextran (Ac-Dex) nanoparticles were synthesized to contain PTX and/or imatinib. These particles were shown to have sizes appropriate for systemic delivery (approximately 250 nm) and exhibited smooth, round morphology.
All particle systems exhibited favorable homogeneity as evidenced by low polydispersity index values (less than 0.20) and were nearly neutral in charge with zeta potentials near zero. Both PTX and imatinib were successfully loaded into the nanoparticles with 50 and 14 weight % loading, respectively. 80% of iRGD was conjugated to the nanoparticles and the peptide showed affinity for A549 lung adenocarcinoma cells in vitro. A549 multicellular spheroids were generated to allow for evaluation of the newly developed therapeutics. Finally, in vitro analysis using A549 cells showed that the drug-loaded nanoparticles were more effective at killing cells than pure drugs. Overall, this work shows the promise of Ac-Dex drug-loaded nanoparticles conjugated with iRGD to enhance the treatment of lung cancer.   The optimization and improvement of cancer therapeutics is currently an important objective of pharmaceutical research since there are still many problems faced in the treatment of many types of cancer including lung, glioblastoma, and pancreatic cancers, which have extremely poor treatment success rates (Sneed 1998 In regards to chemotherapeutics, there are several drug classes used in combination with one another as the first-line therapies for many types of NSCLC (Kelly 2001, Schiller 2002. Of these drugs, there are many FDA-approved compounds currently used clinically for the treatment of NSCLC including paclitaxel (Jinhyang 2012) and cisplatin (Zhang 2011). In addition, there are many drugs under investigation for the treatment of NSCLC such as imatinib (Zhang 2003), everolimus (Kochanski 2012), and artesunate (Zhao 2011). While these particular drugs are not yet widely used in the treatment of NSCLC, they are currently approved for the treatment of other types of cancer.

LIST OF TABLES
The ability of chemotherapeutics to kill cancer cells depends on their ability to halt cell division. Usually, these types of drugs work by damaging the RNA or DNA that tells the cell how to copy itself in division (Vermeulen 2003). If the cells are 5 unable to divide, they subsequently die. The faster the cells are able to divide, the more likely it is that chemotherapy will kill these cells, causing the tumor to shrink. This is the main tenant behind how chemotherapeutics work in that they attack rapidly growing cancer cells while sparing the majority of the body's healthy tissues.
However, this means that these therapeutics will also attack rapidly growing cells in other parts of the body including hair follicles and gastrointestinal tract.
Chemotherapeutics ultimately induce apoptosis (self-death or cell suicide). Drugs that disrupt mitotic progression, which are commonly referred to as antimitotics, are used extensively for the treatment of cancer. Paclitaxel (Taxol®, Bristol-Myers Squibb Co., Princeton, New Jersey), which belongs to this class of chemotherapy drugs (Marupudi 2007), was originally derived from the bark of Taxus brevifolia (pacific yew tree). The structure of PTX can be seen in Figure 1. PTX binds to polymerized tubulin in cells, thereby preventing microtubule formation and promoting microtubule stabilization against disassembly. This in turn inhibits mitosis, and therefore prevents cancer growth.
Imatinib mesylate (also known as simply imatinib, STI571, or Gleevec from Novartis Pharmaceutical Inc.), is a recently FDA-approved drug used for the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. The structure of imatinib can be seen in Figure 2. Imatinib is an adenosine triphosphate (ATP) analogue, and it competitively binds to and inhibits the oncogene BCR-ABL tyrosine kinase, which results from the chromosomal translocation (Zhang 2003). While traditional cytotoxic cancer treatments such as chemotherapy or radiation therapy kill all dividing cells, imatinib acts on a molecular target by a mechanism that is more  specific to cancer cells (Timeus 2012). Therefore, it is advantageous to use imatinib for the treatment of cancer because it is more specific to damaging cancer cells, thereby sparing healthy tissue. In particular, imatinib mesylate (IM) has been demonstrated to inhibit the growth and proliferation of gastrointestinal stromal tumors (GIST), and to induce the complete or partial clinical remission in GIST patients. In addition, IM was showed to inhibit the growth of dermatofibrosarcoma protuberans (DFSP) through inhibiting the platelet-derived growth factor (PDGF) receptor (Zhang 2003 it has been demonstrated that A549 cells overexpress PDGF receptor alpha (PDGFR α), one of the known potential targets for imatinib. The inhibitory effect of imatinib on A549 cells is likely mediated through inhibition of this receptor via phosphorylation (Zhang 2003). Overall, these results show the promise that IM has to selectively target and treat a wide variety of tumors.
Despite the widespread use of the aforementioned therapeutics for the treatment of cancer, as with other types of chemotherapeutics, they often face challenges in therapeutic efficacy due to limitations in systemic delivery. In particular, the delivery of paclitaxel faces many challenges due to its lipophilic nature and high protein affinity, which ultimately results in low bioavailability due to its very minimal solubility in water. Also, while the addition of Cremophor EL (a type of polyethoxylated castor oil) and ethanol in the intravenous Taxol® formulation is used to increase the solubility of PTX, these additives often cause severe side effects in many patients (Carvalho 2011).
It has been shown that drug solubilization, an increase in tumor accumulation, and toxicity attenuation of drugs can be enhanced via the use of nanoparticles for the delivery of anti-cancer agents. For example, single-agent paclitaxel plays a central role in the treatment of advanced NSCLC. As mentioned previously, the use of this drug is limited, however, by its poor solubility and by the toxicities associated with additives in the formulation Taxol®. Abraxane is an FDA-approved therapeutic that is comprised of a paclitaxel protein (albumin)-bound nanoparticle system delivered to patients via an injectable suspension. These nanoparticles were developed by American BioScience, Inc. (Santa Monica, California) and are Cremophor EL-and ethanol-free. That nanoparticles are approximately 130 nm size and have been designed to address the solubility limitations of traditional paclitaxel (Green 2006).
In addition, paclitaxel-loaded poly(ethylene glycol) and poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles have been developed where this new system is more cytotoxic on Hela cells than Taxol® (Goodman 2008). In contrast to Cremophor EL, no cytotoxicity of the polymers that make up these nanoparticles was observed (Danhier 2009). In regards to IM, imatinib mesylate has also been successfully loaded into PEG-PLGA nanoparticles as an model system for successful intracellular delivery of molecular targeting drugs in excised veins (Kimura 2008). Similarly, imatinib mesylate-chitosan poly(ε-caprolactone) (PCL) nanoparticles have been investigated for their potential in enhancing controlled release and improving chemotherapeutic efficiency of IM (Cortese 2014). In regards to tandem delivery vehicles, dual-drug loaded nanoparticles containing PTX and gemcitabine have been used against human pancreatic cancer cells and their cytotoxicity was significantly improved compared to the free drugs (Aryal 2010). A study from the Acharya lab demonstrated that dual-drug loaded nanoparticles work in a synergistic manner lowering the dose of administration and time of action of the drugs, ensuring that both drugs reach the targeted cells specifically and maximize cytotoxicity while minimizing the chances of cell resistance to any one drug (Acharya 2011). Current treatment, however, revolves around the combined delivery of multiple therapeutics, and this is not easily achieved with single-agent nanoparticle systems demonstrating the need for a therapeutic capable of tandem delivery of chemotherapeutics. Overall, these studies show the feasibility in using drug-loaded nanoparticles to enhance the treatment potential of currently available anticancer therapeutics.

Tumor-Penetrating Peptides
There are currently many drug delivery systems in development for the treatment of lung cancer and some of them have achieved moderate progress and have brought about the considerable development in this area of research. In fact, several targeted drug and gene delivery systems have been developed to treat NSCLC more effectively. Nowadays, much attention is being focused on nanotechnology, where this refers to the interactions of molecular components and engineered materials at the nanoscale.
Nanomaterials are typically clusters of atoms, molecules, and molecular fragments formed into incredibly small particulates. Nanometer-sized particles have novel optical, electronic, and structural properties that are not always present in individual molecules or bulk solids (Sajja 2009). The concept of nanoscale devices has led to the development of biodegradable self-assembled nanoparticles, which have been engineered for the targeted delivery of anticancer drugs and imaging contrast agents.
Nanoconstructs such as these can serve as customizable, targeted drug delivery vehicles capable of ferrying large doses of chemotherapeutic agents or therapeutic genes into malignant cells while sparing healthy cells. Such ''smart'' multifunctional nanodevices hold out the possibility of radically changing the practice of oncology, allowing easy detection and then followed by effective targeted therapeutics at the earliest stages of the disease (Sinha 2006). In particular, polymeric nanoparticles (PNPs) are defined as particulate dispersions or solid particles with size in the range of 10 -1000 nm. There has been considerable research interest in the area of drug delivery using particulate delivery systems as carriers for small and large molecules.
The techniques for the preparation of nanoparticles depend on the particular application and or drug to be loaded into the nanoparticle system. A mini-review of several common methods for the preparation of nanoparticles from dispersions of 12 preformed polymer can be seen in Table 2 and methods for preparation of nanoparticles from polymerization of monomers in Table 3.
13 Table 2: Review of the common methods used for the preparation of nanoparticles.

Preparation Method Preparation Details
Emulsion Emulsion polymerization is one of the fastest methods for nanoparticle preparation and is readily scalable. The method is classified into two categories, With the increased use of nanoparticles for drug delivery applications, it has been noted that there are many advantages in using these systems over traditional systemically delivered pure drug systems (Kayser 2005, Abhilash 2010). Such advantages in the use of polymeric nanoparticle systems include, but are not limited to the following:  The increased stability of any volatile pharmaceutical agents present.
 The fabrication of large quantities of particles is easy and cheaply done using a multitude of methods.
 A significant improvement over traditional oral and intravenous methods of administration in terms of efficiency and efficacy is often seen.
 There is delivery of a higher concentration of a pharmaceutical agent to a desired location.
 Modified drug release allows for tailoring of drug delivery.
 They can be applied to a wide variety of drug delivery fields including cancer therapy, delivery of vaccines, contraceptives, and targeted antibiotics.
 The can be easily incorporated into other activities related to drug delivery, including tissue engineering.
The enhanced permeability and retention (EPR) effect involves the extravasation of nanoparticles from "leaky" tumor vasculature into tumor tissue. While the EPR effect has served as the key rationale for using nanoparticles to treat solid tumors, it often does not enable uniform delivery of these particles to all regions of tumor.
20 Furthermore, while ligands that target cancer cells can often increase the intracellular concentration and cytotoxicity of targeted nanoparticles, the intratumoral penetration is not improved significantly compared with non-targeted nanoparticles (Jain 2010).
This is due to multiple factors including ineffective targeting ligands, the tumor-blood vessel wall barrier, slow diffusion into the interstitium of tumors, and physiological barriers of the tumor. These barriers include the lack of functional lymphatic vessels, elevated interstitial fluid pressure, and dense extracellular matrix (Jain 1990).
Tumor-penetrating peptides show great promise in overcoming many barriers that prevent the accumulation and penetration of nanoparticles into the avascular region of tumors. iRGD (internalizing RGD, CRGDKDPDC), a cyclic peptide, has been shown to effectively target and penetrate both in vitro three-dimensional multicellular spheroids and in vivo animal model tumors when conjugated to drugs or nanoparticles (Sugahara 2010). The peptide iRGD initially accumulates at the surface of αν integrin-expressing cells and tissue in tumors. This is advantageous in that integrins are overexpressed in many types of tumors and tumor-associated vasculature and tissue while being minimally expressed in healthy tissue (Waite 2009, Desgrosellier 2010. Once iRGD reaches the integrin target, it is cleaved by cell surface-associated proteases to expose the C terminus and CandR element which then mediates binding to neuropilin-1, resulting in penetration of the cancerous cells and tissues as seen in Figure 3. iRGD has been used effectively with many new and already FDA-approved nanoparticle systems and drugs such as doxorubicin, Abraxane®, Doxil® and trastuzumab to enhance tumor penetration and treatment efficacy in in vivo models ). In addition, it enhances tumor penetration when co-administered

22
with drugs and particles.
The contribution of the therapeutic nanoparticle systems developed in this current research lies in the production of a system that will be easier to deliver to patients by combining multiple agents into one platform. Furthermore, it will allow not only for the direct targeting of anticancer agents to lung tumors but will induce penetration of the nanoparticle into tumor tissue, which can potentially allow for a lower dose of the drugs to be used due to increased specificity (Simberg 2007. The potential to lower the dosage of chemotherapeutic drugs using a nanoparticle drug delivery system could result in a treatment that is both safer for patients and easier to tolerate, especially for those that are not ideal candidates for aggressive treatment. Both the elderly and patients with morbidities such as chronic obstructive pulmonary disease (COPD) could benefit greatly from this type of treatment since they are usually neither eligible for surgery nor healthy enough for chemotherapy (Schroedl 2012). Since 50 -80% of lung cancer patients have COPD (Young 2009), there is a significant portion of the population that could benefit from a more targeted, safer treatment option.

Multicellular Spheroids (MCS) in Cancer Treatment Evaluation
Determining whether or not nanoparticles will penetrate the tumor microenvironment is often difficult and while cell uptake studies are most commonly used to show this potential, they bear minimal physiological resemblance to tumors. MCS can mimic avascular in vivo tumors in that they have diffusion limitations for many molecules, including oxygen, and this inefficient mass transport leads to metabolic waste accumulation inside the MCS so that they display a layer-like structure comprised of a necrotic core surrounded by a viable rim of cells (Lin 2008).
It is usually the center of tumors that exhibit a hostile microenvironmrnt, as these regions harbor the most aggressive tumor cells, which will regenerate if they are not eliminated. MCS have been effectively used to study many types of therapeutics including nanoparticles, chemotherapeutics, and radiation where the MCS show significant differences in reponse in comparison to 2D cell monolayers (Goodman 2007, Goodman 2008. This illustrates imparts the importance of these systems in optimizing drug and nanoparticle systems for systemic delivery to tumors.
24 Figure 4: Schematic depicting the formation of spheroids for loose cell aggregates into a compact spheroid following a brief delay period in formation.

Acetalated Dextran (Ac-Dex) as a Novel Drug Delivery Platform
Acetalated dextran (Ac-Dex) is a newly developed polymer in which pendant acetal groups are reacted onto hydroxyl groups present on the backbone of dextran to give this polymeric compound unique characteristics. The reaction of dextran with 2-methoxypropene leads to the formation of both cyclic and acyclic acetals, causing a switch in solubility and providing a trigger for acid-mediated degradation ). Cyclic and acyclic acetals have completely different rates of hydrolysis (Fife 1965) and the amount of each present can be tailored by the extent of reaction allowing for the modulation of degradation rates from hour to weeks (Meenach 2012).
In addition to allowing for tunable degradation, Ac-Dex has solubility properties opposite of those of parent dextran, namely that it is soluble in organic solvents and insoluble in water allowing for the processing of Ac-Dex into nanoparticles via standard emulsion techniques . Furthermore, Ac-Dex has pH neutral degradation by-products (parent dextran and small amount of acetone and methanol) and is acid sensitive. At low pH Ac-Dex degradation occurs more quickly whereas at higher pH, the polymer degrades more slowly. In vitro assays have indicated that Ac-Dex and its degradation by-products are as safe as the FDA-approved polymer poly(lactic-co-glycolic acid) (PLGA) , which is commonly used in polymeric nanoparticle formulations. The use of Ac-Dex for the tandem delivery of agents significantly advances the development of a targeted biodegradable nanocarrier in that the carrier can be made to degrade fully within a day or up to a week without harmful polymer accumulation or degradation by-products. Thus far, Ac-Dex has been used in particle formulations for applications in immunology/vaccines , (Kanthamneni 2012, and pulmonary therapeutics (Meenach 2012).

Research Aims and Objectives
The overall aim of this project was to develop and optimize acetalated dextran-based peptide-conjugated polymer nanoparticles, which can deliver anti-cancer agents in tandem to allow for enhanced targeting and treatment of non-small cell lung cancer. The specific research for this project include:  To synthesize acetalated dextran-based iRGD-conjugated nanoparticles loaded with paclitaxel and/or imatinib mesylate.
 To evaluate and optimize the physicochemical properties the peptide-conjugated Ac-Dex nanoparticles.
 To assess the efficiency of the proposed nanoparticle system in the treatment of non-small cell lung cancer using in vitro methods.

Materials
Phosphate buffered saline ( (Burlingame, CA, USA). All materials were stored in the conditions recommended by the manufacturer and were used as received.

Synthesis and Characterization of Acetalated Dextran (Ac-Dex)
Ac-Dex was synthesized by the reaction of dextran with 2-methoxypropene as seen in Figure 5. 1 g of lyophilized dextran (9,000 -11,000 MW) was mixed well with 15.5 mg pyridinium p-toluenesulfonate in 10 mL of anhydrous DMSO to allow for complete dissolution of the dry components. The reaction was initiated by adding 1 mL of 2-methyoxypropene, which added the acetal groups (both cyclic and acyclic) to the parent dextran. The reaction proceeded for five minutes to produce fast degrading Ac-Dex ). The reaction was then quenched with 1 mL of triethylamine (TEA), followed by precipitation in basic water (pH 9.5), isolation via filtration, and lyophilization. Basic water was made by mixing TEA in deionized water for a final pH value of 9.5. This solution prevents the early degradation of pH-sensitive Ac-Dex.

30
produces one acetone and one methanol molecule. Therefore, from the relative ratio of the peaks from acetone, methanol, and the cyclic to acyclic ratio of acetal substitution and degrees of hydroxyl substitution per 100 glucose could be determined using 1 H-NMR ). The cyclic to acyclic acetal ratio will be further referred to as the CAC. After centrifugation, the supernatant solution was decanted and 1 mL of basic water (pH 9.5) was added to each tube. The resulting particle pellets were then resuspended in the basic water using a pipette and water bath sonication, as needed.

Nanoparticles
The remainder of the tubes with the resuspended nanoparticles was filled with basic water and this centrifugation and washing step was repeated until the particles had been washed three times. After the final decantation, the particles were suspended in 1 mL of basic water, and this solution was then transferred to a 5 mL tube. A further 3 mL of basic water was used to rinse the centrifuge tubes and this wash solution was added to the 5 mL tube for a final volume of 5 mL. The particle solution was then put in the freezer for one hour followed by lyophilization for 24 hours. A schematic of this process can be found in Figure 6.
The mean size (diameter), polydispersity index (PDI), and zeta potential of the particles was determined using a Malvern Zetasizer Nano ZS capable of dynamic light scattering (DLS) and zeta potential measurement. The shape and surface morphology  Figure   A1 and Figure A2, respectively) were generated using UPLC as described above.

34
These calibration curves are present in Appendix A. From this analysis the encapsulation efficiency and drug loading of the nanoparticles were evaluated as: Furthermore, the percent of drug released was evaluated from: where M t is the mass of the drug at time point (t) and M is the mass of drug in sample.
Thermal analysis and phase transition measurements were carried out using a TA Q10 differential scanning calorimetry (DSC) system (TA Instruments, New Castle, DE, USA) with TA Instruments Explorer software and DSC refrigerated cooling accessory. 1 to 5 mg of sample was weighed into hermetic anodized aluminum DSC pans and was sealed hermetically sealed. Dry nitrogen gas was used as the purging gas at 50 mL/min. The heating range during DSC analysis was 0 to 300 °C at a heating scan rate of 10.00 °C/min. After 48 hours, a resazurin assay was used to assess the cell viability of the cells.

Lung carcinoma (A549) cancer cells (obtained from American Type
Resazurin is converted to highly fluorescent resorufin by cells that are alive. 20 L of 20 mM resazurin was added to each well containing cells and the well plates were As seen above, these results were reported as the ratio of the fluorescence intensity of the resorufin generated from live cells for a given sample exposed to treatment in comparison to the fluorescence intensity generated from live cells not To evaluate the potential uptake of nanoparticles into lung cancer cells, A549 cells were seeded at 125,000 cells/mL (300 μL/well) in 8 well chamber slides and were placed in an incubator for 24 hours to allow time for cell attachment. The cells were then exposed to 1 mg/mL of fluorescently-labeled drug-loaded nanoparticles in media for 24 hours at 37 °C. These nanoparticle systems were 0.1% PTX-loaded and 10% imatinib-loaded nanoparticles with the addition of 2 weight % fluorescein isothiocyanate (FITC) added to the organic phase during nanoparticle synthesis to allow for fluorescent imaging of the particles. After the cells were exposed to the nanoparticle/media solutions (up to 24 hours), the particle solutions were removed and the cells were washed twice with 200 mM glycine to remove any unbound particles, followed by an additional wash with PBS. The cells were then stained with 750 nM Lysotracker Red and fixed with hardset Vectashield containing DAPI prior to imaging with a Nikon Eclipse E600 upright microscope.
The ability of A549 cells to form spheroids and their growth with time was evaluated using brightfield microscopy (Nikon Diaphot TMD). The spheroids were imaged immediately after centrifugation and then each day for up to 5 days. The size of the spheroids was analyzed using ImageJ software (Abramoff 2004).

iRGD Conjugation and Tumor-Penetration Efficacy of Nanoparticles
Two types of iRGD were used for the studies described in this research. The first type of iRGD was peptide with no fluorescent tag and extra cysteine (G) residues that was synthesized, purified (99 % purity), and characterized by Rakesh Tiwari, a collaborator in the Department of Biomedical and Pharmaceutical Sciences at the University of Rhode Island (Kingston, RI). The final compound (NH 2 -CGGGCRGDKGPDCG-CONH 2 ) was an alkoxyamine-functionalized peptide that could be conjugated to Ac-Dex nanoparticles owing to the ability of polysaccharides to form stable oxime conjugates with alkoxyamine-bearing molecules ). The second type of iRGD use in the described studies was CRGDKGPDC labeled with a TAMRA fluorophore to allow for easy detection of the iRGD in solution. This compound was generously donated by Anastasia Kruse, a collaborator in the Department of Chemical Engineering at the University of Kentucky (Lexington, KY). Figure 7, iRGD-conjugated nanoparticles were prepared by mixing 5 mg of the alkoxyamine-functionalized iRGD with 1 mg of Ac-Dex nanoparticles in 1 mL PBS. First, 5 mg of iRGD and 1 mg of nanoparticles was measured into 1 mL of PBS and was then sonicated in a water bath for 15 minutes.

As depicted in
The samples were then put on a shaker at 37C and 100 rpm for 2 days. After 2 days, the samples were centrifuged at 12,000 rcf (10629 rpm) for 15 minutes and the supernatant was removed. The particles were washed twice with 1 mL fresh PBS and then resuspended using sonication. 100 µL/well of particle solution was added to each

Characterization of Acetalated Dextran (Ac-Dex)
Because of the significant differences in the rates of hydrolysis between the acyclic and cyclic acetals present on Ac-Dex, it is important to know the cyclic acetal coverage (CAC) of Ac-Dex to ensure that the degradation will be appropriate for the given application. The relative proportion of cyclic and acyclic acetals was determined by comparing the relative concentrations of acetone and methanol present during 1 H-NMR analysis. This is possible since the hydrolysis of one acyclic acetal yields one molecule each of acetone and methanol, whereas the hydrolysis of one cyclic acetal yields only a single acetone molecule . The CAC was then determined via the ratio of acetone to methanol by comparing the integration of the acetone peak (2.08 ppm) to the methanol peak (3.34 ppm) from 1 H-NMR analysis. All integrations were normalized to the number of protons on each molecule. The calculation protocol for this method can be found in Appendix B.
Analysis of Ac-Dex formed after a 5-minute reaction period was completed via 1 H-NMR (see Figure 8). These results showed that the cyclic acetal coverage (CAC) of the fast degrading Ac-Dex was approximately 70%. In addition, the conversion of hydroxyl groups on the parent dextran to acetal group was at least 80% for all batches

45
of Ac-Dex, indicating good coverage of acetals on the polymer backbone. The reported CAC value is appropriate for the desired application as Ac-Dex with this property is known to degrade with a few days , Meenach 2012, which is appropriate for the application of nanoparticles to tumors.

Characterization of Drug-Loaded Ac-Dex Nanoparticles
The size (diameter), polydispersity index (PDI), zeta potential, encapsulation efficiency, and drug loading of the nanoparticles was evaluated to show the feasibility and efficacy of using the emulsion/solvent evaporation method for the generation of chemotherapeutic nanoparticles. The size of both blank (no drug) and drug-loaded Ac-Dex nanoparticles was evaluated using dynamic light scattering (DLS) and the results are depicted in Table 4. The particles sizes range from 232 to 282 nm, which is suitable for nanoparticles used in the applications for the systemic delivery of nanoparticles and cancer treatment via tumor penetration and controlled delivery.
Nanoparticle formulation and surface morphologies were visualized and analyzed via scanning electron microscopy (SEM) as seen in Figure 9. From these images it can be seen that the nanoparticles are smooth and spherical, which is appropriate for the indicated application. From these images it can be estimated that the diameters of both the blank and drug-loaded nanoparticles range from 200 to 300 nm, confirming the results gained from the DLS analysis. Finally, no differences were seen between systems with paclitaxel (PTX), imatinib mesylate (IM), and blank nanoparticles regarding their morphology, indicating the loading of the drug had little effect on these 46  characteristics in comparison to blank particles without drug.
The polydispersity index (PDI) values from Table 4 were approximately 0.20, indicating that the nanoparticles, regardless of the presence or type of drug in the nanoparticle systems. In addition, the zeta potential values of the nanoparticles ranged from -2.43 to -0.30 mV, indicating that the particle systems are all nearly neutral , with drug loading have no effect on this parameter.
The encapsulation efficiency of the nanoparticles was 50 % for 0.1% PTX nanoparticles and 14 % for 10% imatinib nanoparticles. This ultimately resulted in an actual drug loading of 0.52 g of PTX per mg of nanoparticles and 14 g of imatinib per mg of nanoparticles. Overall, these results indicate that both PTX and imatinib can be successfully loaded into Ac-Dex based nanoparticle systems using the emulsion/solvent evaporation particle formation method. In addition, it seems that there is some effect on drug loading due to either the type of drug loaded or the amount of drug loaded into the nanoparticle systems. This could be due to the hydrophobicity or lipophilicity of the drugs present in the nanoparticle systems. Figure 10 shows the results from differential scanning calorimetry (DSC) for the nanoparticle systems and raw components that made up the nanoparticles. While, the nanoparticles were evaluated a large temperature range, these results ultimately show that the nanoparticles are stable within a normal temperature range that nanoparticles would be exposed to during synthesis, storage, and administration (4 to 100°C).
The release of paclitaxel and imatinib from acetalated dextran nanoparticles was evaluated over 48 hours as seen in Figure 11 (for paclitaxel nanoparticles) and Overall, these results demonstrate the feasibility in synthesizing PTX and IM-loaded nanoparticles with Ac-Dex as the polymer. The systems ultimately exhibit the desired morphological, size, charge, and shape necessary for systemic delivery to tumors and are stable at the desired working temperatures. Furthermore, the nanoparticles exhibit the ability to control the release of either PTX or IM for an extended amount of time, which can enhance the efficacy of using these systems in the treatment of lung cancer.

In Vitro Biological Characterization
The effect of raw paclitaxel, paclitaxel-loaded nanoparticles, and blank nanoparticles on the viability of A549 lung adenocarcinoma cells was evaluated 48 hours after drug and/or nanoparticle exposure. The effect of raw PTX, PTX-loaded nanoparticles and raw Imatinib on A549 cells can be seen in Figure 13 and 14. For both the pure drug and PTX nanoparticles, the cell viability decreased with increasing drug concentration as expected. Furthermore, the relative viability of A549 cells exposed to PTX-loaded nanoparticles was lower than that for pure PTX. This  values, were the IC50 value for raw PTX was 0.2299 µM and for PTX NPs was 0.0695 µM. This ultimately indicates that the PTX-loaded nanoparticles are more effective in killing lung cancer cells than pure PTX, even without iRGD conjugation.
And the IC50 of raw Imatinib was 4.0993 µM .
The effect of blank nanoparticles on A549 cells was also evaluated. The amount of nanoparticles loaded into media for exposure to the cells was determined by using the largest mass of nanoparticles needed for the highest concentration of PTX. The relative viability of cells exposed to blank nanoparticles was 96 %, indicating that the nanoparticle exert minimal to no cytotoxic effects on A549 cells. This, in turn, demonstrates that they should be safe for administration of anticancer therapeutics.
The potential for Ac-Dex nanoparticles to be uptaken into A549 cells was evaluated. For these initial studies, FITC-loaded nanoparticles with no drug were evaluated by exposing the cells to nanoparticles for 5 hours. Figure 14 shows that after 5 hours the A549 cells exhibited very minimal uptake of FITC-loaded nanoparticles. This could potentially be attributed to the short exposure time or the lack of peptide targeting for these nanoparticles since no iRGD was conjugated onto the nanoparticle surfaces.

Three-Dimensional Multicellular Spheroid Evaluation
Three-dimensional (3D) multicellular spheroids (MCS) comprised of A549 lung cancer cells were successfully formed using a modified centrifugation technique. In this technique, cells in a small amount of collagen were seeded into non-adherent round bottom 96 well plates followed by mild centrifugation. As shown in Figure 15, The size of the spheroids was analyzed by using the brightfield images and ImageJ software. As seen in Figure 16, the size of the spheroids increased with culture time and the spheroids reached a diameter of approximately 560 m after 5 days. Overall, these results indicate the successful formation of A549 multicellular spheroids where these systems will be used in future studies relating the tumor-penetrating nanoparticles.

iRGD-Nanoparticle Conjugation and Affinity to A549 Cells
The amount of iRGD capable of conjugating to acetalated dextran nanoparticles was determined using the fluorescamine assay. These results indicated that approximately 5.618 x 10 4 molecules of iRGD were conjugated to each nanoparticle and the iRGD coverage is 0.287 iRGD/nm 2 . Overall, these results show that acetalated dextran nanoparticles can be produced that will be capable of tumor homing and penetration through the iRGD tumor penetrating mechanism.
The affinity of iRGD to A549 cells was evaluated by exposing these cells to differing concentrations of TAMRA-conjugated iRGD for different times (up to 24 hours). These results are indicated in Figure 17, which showed that iRGD interacts with and likely penetrates A549 lung adenocarcinoma cells. In this figure, fluorescence intensity of the TAMRA-iRGD attached to the cells is compared to the iRGD concentration and exposure time. After 3 hours, the fluorescence intensity of the samples was the highest regardless of iRGD solution concentration. This indicates that this is the optimal time period for A549 cells to interact with and uptake iRGD, which will be useful in designing future in vitro experiments with respect to exposure time.
Overall, the fluorescence intensity of the samples increased with increasing TAMRA-iRGD concentration. This is expected because there is a linear relationship between the concentrations of iRGD in serum-free media with respect to fluorescence intensity as shown by the calibration curve in Figure A3 (see Appendix A). Overall, these studies show the feasibility in using iRGD-conjugated nanoparticles to target A549 lung adenocarcinoma cells for the enhanced treatment of lung cancer.

Conclusions
Two kinds of drug-loaded nanoparticles were successfully synthesized using the polymer acetalated dextran in this study. These systems were comprised of 0.1% paclitaxel (PTX) and 10% imatinib-loaded nanoparticles. Overall, their physical and chemical characteristics have been evaluated. The developed systems exhibited favorable size (diameter), polydispersity in relation to size, and zeta potential to allow for their use in the systemic delivery of anticancer therapeutics. Both the encapsulation efficiency and drug loading were also favorable for the application of these nanoparticles for potential the treatment of lung cancer.
In vitro cell studies showed that drug-loaded nanoparticles increases the efficacy of chemotherapeutic drugs (in particular paclitaxel) for A549 lung carcinoma cells. It is possible that chemotherapeutic drug-loaded Ac-Dex nanoparticles could be applied in vivo to treat non-small cell lung cancer while reducing the toxic side effects of the free drug normally delivered systemically.
For this application, drug-loaded Ac-Dex nanoparticles could potentially be actively targeted to non-small cell lung cancer tumors and vasculature and then penetrate into the tumors cells with the help of tumor-penetrating peptide, iRGD. This intracellular environment would then trigger the intracellular burst release of the drug payload due to the acid-sensitivity of the Ac-Dex polymer with minimal drug released 63 in extracellular pH-neutral environments. The results of the described study indicate that iRGD can be successfully conjugated to Ac-Dex nanoparticles and that iRGD also shows affinity for A549 cells that are known to overexpress integrins that allow active targeting of the nanoparticle systems. Overall, these drug-loaded nanoparticles may have the ability to treat non-small lung cancer in addition to numerous other types of cancer.

Future Work
The work described in this thesis is hopefully just the beginning regarding the investigation of tandem delivery of agents using nanoparticles system for the treatment of non-small cell lung cancer. Future work to build upon this project may include, but is not limited to:  Optimization of nanoparticles size and encapsulation efficiency to achieve a better effect during in vitro studies.
 Investigation of the methods of loading two drugs into one nanoparticle system to reduce side effects and allow for dual delivery.
 Optimizing and evaluating the conjugation of iRGD onto blank and drug-loaded nanoparticle systems.
 Investigation into the ability of iRGD-conjugated nanoparticles to increase the efficacy of non-iRGD nanoparticles against A549 cells on both 2D monolayer and 3D spheroid cultures.

64
 Evaluating the ability of iRGD-conjugated nanoparticles to penetrate 3D multicellular spheroids.
 Evaluating the systemic efficacy of iRGD-conjugated Ac-Dex nanoparticles in vivo in both non-small cell lung cancer cells and tissues.
 Incorporation of iRGD-conjugated, drug-loaded nanoparticles with radiation treatment to increase their therapeutic effect.

APPENDIX A CALIBRATION CURVES
As described in Chapter 2 (Materials and Methods), calibration curves for paclitaxel (PTX) and imatinib mesylate (IM) were generating using ultra performance liquid chromatography (UPLC). As seen in Figure A1, a plot of the peak area due to the detection of PTX compared to its concentration was generated. This figure shows a lower detection limit of 10 g/ml and upper detection limit of 80 g/ml in the mobile phase for PTX. The R-squared value of 0.9999 shows very good fit for the described data. A plot of peak area due to the detection of IM compared to its concentration can be found in Figure A2. This figure shows a lower detection limit of 10 g/ml and upper detection limit of 100 g/ml for imatinib mesylate in mobile phase with a favorable R-squared value of 0.9944.
TAMRA-iRGD calibration curves were generated using fluorescence detection of a plate reader as described in Chapter 2 (Materials and Methods) as seen in Figure   A3. For this detection, the lower limit was 0.0005 g/ml and the upper limit was 0.05 g/ml with a favorable R-squared value of 0.9. Finally an iRGD-fluorescamine calibration curve was generated using fluorescence microscopy. As seen in Figure A4, the lower limit was 0.1 mg/ml iRGD and the upper limit was 0.5 mg/ml with a favorable R-squared value of 0.9716.

APPENDIX B 1 H-NMR CALCULATIONS FOR AC-DEX
The cyclic acetal coverage (CAC) of Ac-Dex was determined using 1 H-NMR as described in Section 2.2 (Synthesis and Characterization of Acetalated Dextran (Ac-Dex)). Using the software available on the equipment, the areas of characteristic peaks of several compounds were calculated. These included the peak areas for the hydrogen rings present in dextran (3.4 to 4.0 ppm, three peaks total), methanol (3.36 ppm), and acetone (2.08 ppm). First, a normalization factor was calculated for the dextran peaks: where 2 is the number of protons available on each molecule. Next the amount of methanol (MeOH) and acetone (Ace) present in the samples was calculated using the following equations: where NF is the normalization factor. NF was multiplied by 3 for the methanol samples and by 6 for the acetone samples owing to the number of hydrogen molecules present in these compounds. Finally, the cyclic acetal coverage (CAC) was calculated to be: The extent of -OH hydrolysis was also calculated via 1 H-NMR analysis: = − = Two of the three and one of three hydroxyls on each glucose repeat unit can be used to form a single cyclic acetal and acyclic acetal, respectively. The number of -OH groups converted per glucose (-OH Conv) was calculated as: The percent acetalation ratio was then calculated as: 71 APPENDIX C

CALCULATIONS FOR DETERMINING IRGD COVERAGE
The number of iRGD molecules present on the surface of the Ac-Dex nanoparticles was determined using the fluorescamine assay. Fluorescamine reacts with primary amines on iRGD to allow for quantification of the amount of iRGD present in solution. As seen in Figure 22, there is a linear relationship between the fluorescence intensity of fluorescamine and the concentration of iRGD in PBS.
Therefore, the amount of iRGD present on nanoparticles and the iRGD coverage per nanoparticle can be calculated from the following calculations. From the fluorescamine assay, the mass of iRGD present in the sample (m iRGD ) was 0.623 mg.
From this value, the molecules of iRGD per sample was calculated as:

= •
where MW is the molecular weight of iRGD (1569.71 g/moles) and N A is Avogadro's number (6.02214 x 10 23 molecules/mole). The molecules of iRGD in 1 ml of solution was determined to be 3.969 x 10 16 molecules. The number of nanoparticles present in each sample was determined from the nanoparticle volume, density, and mass per sample. The mass of an individual nanoparticle is calculated as: where V is the volume of a 255 nm nanoparticle (1.413 x 10 7 nm 3 ) and ρ is the density of the nanoparticle (1 g/cm 3 ), which is the density of most polymers. This gives a mass of a single nanoparticle to be 1.413 x 10 -11 mg. The number of nanoparticles in a sample was then calculated from the total mass of nanoparticle (m T = 5 mg) and mass of a single nanoparticle:

# =
where the total number of nanoparticles per sample was 7.065 x 10 11 . The number of iRGD present on each nanoparticle was then calculated from the number of nanoparticle (m n ) and number of iRGD molecules: