Inhalation of aerosolized drugs has become a well-established means of treating localized disease states within the lung. More recently it has been demonstrated that the lung may also be an ideal site for the delivery of peptides and proteins to the systemic circulation, a route which is generally much more acceptable to patients than an injection. However, due in part to the high absorptive capacity of the lung, most medications in aerosol form require inhalation at least 3–4 times a day due to the relatively short duration of resultant clinical effects.

The design and use of biodegradable polymers for the controlled delivery of entrapped drugs is an approach that holds promise for improving the duration and effectiveness of drugs delivered locally or systemically via the lung. Polymer-based drug delivery vehicles can also reduce toxicity due to spikes in drug concentration commonly seen with traditional immediate-release therapeutic aerosols. However, many problems remain to be addressed before a dry powder polymer-based medicinal aerosol product can be realized. Perhaps the most important is the need for a polymer engineered specifically for pulmonary drug delivery, since existing polymers have poor properties for chronic drug administration in the lung.

We are currently engineering novel drug-containing polymeric devices with: (1) degradation times appropriate for pulmonary drug delivery (thereby reducing polymer build-up in the lung upon repeat administration); (2) improved surface chemistries to optimize aerodynamic performance (thereby increasing delivery efficiency); and (3) properties that reduce particle clearance rates in the deep lung (thereby increasing drug delivery duration).

A countless number of drugs could potentially benefit from controlled delivery via the lung, including many hormones, cytokines, asthma medications, insulin, vaccines, genes and more.

Below is the novel polymer we synthesized.

Picture: Cast of human lung.

New advances in biotechnology and molecular biology are generating scores of potent therapeutics that are opening the doors to combat many diseases. With the appropriate delivery vehicle, these therapeutics can be delivered locally to various regions of the body, for example to the lungs to treat cystic fibrosis, or into the blood stream to treat the entire body. Inhalation of therapeutics provides a non-invasive method of delivery that offers a more favorable environment compared to the low pH and high protease levels associated with oral delivery, has an enormous surface area for absorption (~100-140 m2), and has a highly permeable epithelium. Inhaled therapeutics can be delivered locally to the lungs, as well as penetrate into the deep lungs where they are transported across the epithelium into the systemic circulation.

Picture left: In vitro lung model used to characterize particle aerosolization and deposition properties.
Picture below: Metal impaction plates of cascade impactor showing particles with un-optimized surface properties (white) and large, porous particles with improved surface properties that are able to reach the deeper lung (pink).

We are designing drug-containing polymeric microparticles from novel polymers synthesized in our lab (see section 1A) to target specific regions of the lung. A countless number of drugs could potentially benefit from controlled delivery via the lung, including many hormones, cytokines, asthma medications, proteins (such as insulin), vaccines, genes and more. Our goals are to produce aerosol particles that release various drugs in a controlled manner, that aerosolization efficiently into the lungs, and that can avoid the phagocytic clearance mechanisms in the deep lungs that gobble up smaller particles. Specifically, we are interested in the development of large, low density particles. The size of these particles significantly decreases particle-to-particle interactions, thereby increasing the ability for these particles to aerosolize into the lung, and reduces particle clearance rates, allowing them to remain in the lung and deliver drugs for extended periods of time. The use of statistical methods to design experiments allows us to optimize new formulations with a minimal number of experiments.

Additionally, we are developing new methods to characterize these microparticles in vitro. To mimic the in vivo conditions the microparticles will encounter when deposited on the lung surface, we are using cell culture models with which we can then characterize drug release, degradation and dissolution of the microparticles (see section 1D).

Several important barriers must be overcome before efficient gene therapy in the lung can be realized. A gene entering the lung via inhalation will first encounter the fluid lining the lung surfaces. The innate immunity of the lung surfaces, including an adhesive mucus layer in the upper respiratory tract, surfactant proteins that function specifically in host defense, and alveolar macrophages in the deep lung, provides a formidable barrier to gene delivery. Genes successfully traversing the mucosal barrier encounter cellular barriers that must be overcome before protein translation can occur. Intracellular barriers to gene expression include cellular uptake, endosomal release, nuclear localization, nuclear uptake, and gene transcription, which may require vector/DNA unpacking. Naked DNA has been an inefficient method of gene therapy in the lung owing to its poor ability to bypass these barriers.

Respiratory mucus lines the luminal side of the tracheobronchial tree from the entrance of the trachea to the terminal bronchioles, humidifying inspired air and trapping small particles and microorganisms until they can be transported out of the lungs. Since the natural role of airway mucus is to remove particle invaders from the lung, transport of drugs and DNA-carrying nanoparticles has been determined to be very inefficient. An improved understanding of the relationship between mucus properties and particle transport is critical to the design of DNA carriers for delivery in the non-distal airways of the lung. By developing a better understanding of the physiochemical properties that increase nanoparticle transport this barrier may be reduced.

Large and light polymer aerosols may improve the duration and effectiveness of drugs delivered via the lung due to their easy aerosolization, controlled degradation properties, and ability to resist phagocytic clearance mechanisms. However, our ability to optimize new formulations for pulmonary drug delivery has been limited by our inability to closely mimic the conditions the particles encounter in the various regions of the lung.

Figure: Aerosol particle on the lung surface

Conventional "complete immersion" methods used to characterize microparticle water uptake rates, polymer degradation kinetics, and drug diffusion rates may not be relevant for large light particles designed for inhalation due to the extremely thin aqueous layers in the lungs. This problem is especially acute in the alveolar region of the lung, where the average thickness of fluid bathing epithelial cells is only approximately 0.07 µm, or two orders of magnitude smaller than our typical aerosol particle. Therefore, we have developed an in vitro cell culture method, utilizing air-interfaced lung epithelial cells, for characterization of aerosol particulates in the lung. This new model will allow a more relevant and quantitative characterization of therapeutic aerosol particles.

SEM image of polymeric particle on lung epithelial cells

Polycationic polymers have shown significant promise as non-viral gene delivery vectors due in part to their ability to condense DNA plasmids of unlimited size and to safety concerns with viral vectors. However, the safety and efficiency of well studied non-viral vectors, poly(L-lysine) (PLL) and polyethylenimine (PEI), has been limited by their non-degradable nature. For example, it has recently been shown that vector unpacking of DNA may act as a rate-limiting step in transfection with non-degradable polycations. Increasing cationic charge density along a polymer backbone was found to correspond to increasing cell cytotoxicity. Additionally, the dose of polycations is often limited due to the toxicity caused by local regions of high cationic charge that is not readily dispersed with non-degradable polymers. I, This suggests that an optimum charge density exists to balance the need for plasmid condensation with the unwanted cytotoxicity of high charge density systems.

A well-designed biodegradable cationic polymer, would allow exact control over charge density so that the optimum may be found between condensation and toxicity. It should also offer the initial protection of DNA from nucleases of a high-molecular weight polymer, but allow for the eventual controlled dispersion of cationic charge into low molecular weight oligomers and biocompatible monomers as intact plasmid DNA is released.

In our lab, we have designed cationic polymers to degrade and release bound DNA once nuclear transport is successful. We have also been able to maintain control over spacing between cationic groups of polymer achieved during synthesis. This control allows optimization of charge density with respect to: (1) DNA/polymer self-assembly, (2) biomaterial-cell and biomaterial-protein interactions that affect particle transport, and (3) the balance between endosomal escape and cellular toxicity.

Collaborator: Professor John van Zanten, NC State University

Synthetic gene delivery systems have shown considerable promise as an attractive treatment for genetic diseases, but success has been limited compared to viral vectors. In particular, little is known about non-viral vector formation kinetics and the interplay between vector structure and transfection efficiency.

The formation of non-viral vectors is driven primarily by electrostatic interactions and counterion release. DNA molecules are negatively charged and are complexed with positively charged polymers, lipids or proteins. As they are mixed together, they self-assemble into complexes and release their counterions. DNA complexation kinetics influence three physical parameters that have a direct effect on gene delivery and expression efficiency: DNA complex geometric size, molar mass (density) and surface charge. We use dynamic and static light scattering techniques to characterize the size and molecular weight of cation/DNA particles. From the size and molecular weight we can determine the density of the complex. We use laser Doppler anemometry to determine the surface charge density (zeta - potential). The cationic polymers we study include polyethyleneimine (PEI), poly-L-lysine, and biodegradable cationic polymers we have synthesized in our lab.

We have shown that long-term delivery (several months) of DNA/polymer nanocomplexes released from PLGA microspheres has led to transfection efficiencies >1,500-fold that of unencapsulated complexes.

Gene delivery is a complex process with many possible rate-limiting steps. After cellular uptake by endocytosis, the gene carrier must traverse the expansive and molecularly crowded cytoplasm to reach the nucleus. Unfortunately, the biophysical and biological mechanisms underlying the intracellular transport of gene carriers remain largely unknown, which limits our ability to make rational modifications to gene carriers for improved gene delivery.

Many groups lump intracellular transport with nuclear entry and refer to these separate processes as the “nuclear translocation” barrier. Clearly, this is not sufficient to understand the transfection process since very different interactions and biological molecules are involved in the two steps. Nuclear translocation is a formidable bottleneck in gene delivery; however, it is unclear whether this limitation is owed to difficulty in gene carrier transport to the nucleus through the crowded cytoplasm, to the transport through the nuclear membrane, or something else completely. Consequently, our research aims to determine and quantify, both temporally and spatially, the intracellular transport properties of gene nanocarriers in real time.

Inhaled aerosols can be effective therapeutic carriers of drugs and genetic material to the upper airways and bronchi. However, efficient drug transport to the epithelial cells of the lung is limited by the mucosal barrier.

Aerosolized gene carriers must traverse a thick mucus gel layer to reach the respiratory cells of the upper lung.

The mucosal barrier is even more important in cystic fibrosis (CF) patients due to a more dense mucus – a result of poor hydration and high content of actin, serum proteins, DNA, alginate, and rigidifying lipids. The development of advanced gene delivery systems that more effectively cross the mucosal barrier should make gene therapy a more feasible method of treating CF. Toward this goal, it is important to understand the relationship between DNA-carrying nanoparticle physico-chemical properties and their transport rates through CF sputum.

Multiple particle tracking (MPT), which combines fluorescence and video microscopy techniques, is a novel approach to studying the transport of nanoparticles in mucus. Using MPT, we have studied the transport of hundreds of individual particles in CF and synthetic mucus.

Positional data is acquired from the recorded images (600 frames, 30 frames/second – 33 ms temporal resolution) by data acquisition software. This data is then used to determine the time-dependent mean squared displacements.

Characterization of individual bead transport rates allows us to focus on and gather much more information about the properties of the most efficient particles, in addition to the determination of the average behavior of all of the particles. Information gleaned from MPT is being applied to the design of more effective aerosol gene carriers.

Systemic chemotherapy often reduces tumor burden in the central nervous system (CNS), but is rarely effective in completely eliminating the tumor. It has been shown that chemotherapeutic molecules can increase the susceptibility of tumor cells to attack by the immune system by altering the antigenicity of their surface antigens. However, because chemotherapeutic molecules often exert their antitumor effects by preventing cell division, they also cripple the ability of the immune system to respond. As a result, systemic chemotherapy is typically not combined with approaches aimed at stimulating the immune system to fight CNS cancer. We hypothesized that local delivery of chemotherapeutic molecules via polymeric systems in the CNS would minimize damage to the systemic immune system, thereby enhancing the effectiveness of a combination chemo/immunotherapy strategy.

Interleukin-2 (IL-2), a potent stimulator of the immune system, was encapsulated into polymer microspheres by the complex coacervation of gelatin and chondroitin-6-sulfate using a low-temperature, water-based method to preserve the activity of this usually labile macromolecule. Unencapsulated IL-2 has a six minute half-life in vivo, however, we showed that encapsulation allows IL-2 to be localized at the site of delivery for over 14 days. Various chemotherapeutic agents (BCNU, adriamycin, taxol, and 4-HC) were encapsulated in poly[(1,3-bis-carboxyphenoxy) propane-co-sebacic anhydride] 20:80, a synthetic polyanhydride currently used clinically to deliver BCNU to treat brain cancer. The combined intracranial delivery of chemotherapeutic and immunotherapeutic molecules was then tested in a lethal rat primary brain tumor model. Combination therapy had a statistically significant synergistic effect on median survival and on the number of long-term survivors in each case. For example, 43% (3 of 7) of rats treated with polymers loaded with 10 % w/w BCNU and with polymer microspheres containing 2.8 % w/w IL-2 (IL-2 MS) survived an intracranial challenge with a lethal dose of 9L gliosarcoma tumor cells (median survival = 71 days). On the other hand, only 12.5% (1 of 8) rats treated with BCNU alone survived (median survival = 41.5 days), and there were no survivors in groups of rats receiving IL-2 MS alone (median survival = 28 days) or placebo polymers (median survival = 18 days). Histology showed massive tumor necrosis due to local chemotherapy, with infiltration by cells of the immune system only when IL-2 was co-delivered, further supporting a possible role for combined chemo/immunotherapy in the treatment of CNS tumors.

We use transgenic animal models to quantitatively study the interaction of the immune system with brain tumors to understand how CNS tumors avoid elimination by the immune system. Math models of this immune response guide development of advanced synthetic systems capable of long-term cytokine delivery to stimulate T cells to more efficiently fight CNS tumors.

Despite significant advances in neuroimaging, microsurgery, radiation therapy, and chemotherapy, the median survival after diagnosis of a malignant brain tumor is still less than one year. It is expected that over 185,000 people in the United States will be diagnosed with a brain tumor this year, and approximately 13,000 will be malignant. Furthermore, brain tumors are second only to leukemia in causing cancer-related deaths in children. The failure of standard therapies to significantly improve the prognosis of affected patients has focused attention on development of innovative alternative treatments, particularly those aimed at stimulating a tumor-specific immune response.

Advances in molecular biology have provided researchers with several potent stimulators of the immune system called cytokines. Despite the initial promise of cytokine-immunotherapy of brain tumors, there has been limited success in treating established brain tumors with this treatment modality. Consequently, basic research into the mechanisms by which malignant brain tumors evade destruction by the immune system is desperately needed. We are using a novel animal system to study the interaction of the immune system, in particular cells called “helper T cells,” with brain tumors in an attempt to understand how CNS tumors avoid elimination by the immune system. We are concomitantly developing advanced synthetic systems capable of long-term delivery of cytokines in an attempt to stimulate T cells to more efficiently fight CNS tumors.

Antigen specific CD4+ helper T cells are necessary for the generation of potent anti-tumor immunity; however, very little is known about the fate of these T cells during tumor progression in the CNS. We are developing tools that combine immunotherapy and chemotherapy for more effective treatment of brain tumors.

The blood brain barrier limits access of the immune system to the brain; thus, it is important to develop effective tools to increase the immune response against brain tumors. Primary central nervous system (CNS) lymphoma represents the second most common tumor after Kaposi sarcoma and the most frequent brain tumor in patients with the acquired immunodeficiency syndrome (AIDS). While the majority of cancer vaccine research has focused on CD8+ cytotoxic T-lymphocyte response, recent evidence indicates that ineffective anti-tumor immune responses are often the result of deficient CD4+ T helper response.

Antigen specific CD4+ helper T cells are necessary for the generation of potent anti-tumor immunity; however, very little is known about the fate of these T cells during tumor progression in the CNS. We have developed a novel animal system in which an identifiable population of naïve hemagglutinin (HA)-specific CD4+ T cells may be monitored in vivo during the progression of a brain tumor on a model A20HA B lymphoma that has been engineered to express HA antigen. This system will allow us to determine changes in tumor-specific T cell phenotype and function following exposure to HA antigen in a brain tumor-bearing host.

Furthermore, we will use an adoptively transferred, tumor-specific CD4+ T cells to perform detailed mechanistic studies on the priming of tumor-antigen-specific T cells in the CNS in response to the specific vaccination with virus HA-Vac, genetically engineered tumor cell lines that produce IL-2, IL-4 and IL-12 and polymer drug delivery systems. These will allow us to thoroughly investigate the mechanisms by which CNS tumors evade immune destruction and the role controlled cytokine delivery systems may play in overcoming this problem. Specifically, we propose: (i) thoroughly investigate the fate of tumor-antigen-specific CD4+ T cells adoptively transferred to mice at various stages of tumor progression in the CNS and their migration to the brain; (ii) analyze the effect of tumor specific vaccination on state of activation or anergy and migration status adoptively transferred tumor specific CD4+ T cells; (iii) investigate the effect of the sustained delivery of potent cytokines (IL-2, IL-4 and IL-12) and the timing of their administration on the fate of T cells throughout the brain tumor progression; (iv) evaluate the effect of combined delivery HA-Vac, IL-2, IL-4 and IL-12 and chemical drugs (BCNU, carboplatin) on the efficiency of adoptive immunotherapy the brain tumor with HA-specific clonotypic CD4+ T cells; (v) test a hypothesis concerning the specific role of generalized stress reaction and steroid hormones on depletion of immune system in process of brain tumor progression. A detailed understanding of the way the CD4+ T cell response to the brain tumors is modulated by local cytokine delivery should allow the rational development of effective new therapeutic strategies.