Dendrimer Conjugation Enhances Tumor Penetration and Efficacy of Doxorubicin in Extracellular Matrix-Expressing 3D Lung Cancer Models.

Doxorubicin (DOX) is a chemotherapeutic agent broadly used in the treatment of a range of solid tumors. In spite of its high potency, as is the case for many other chemotherapeutic drugs, there are many challenges associated with the use of DOX in clinical oncology. This is particularly true for DOX in the treatment of lung cancer, where in vitro potency is shown to be very high, but low lung distribution and off-target toxicity (particularly cardiotoxicity) restrict its use. Nanocarrier-based drug delivery systems (nanoDDS) have been shown to help alter biodistribution and alleviate off-target toxicity associated with DOX. While significant understanding exists regarding the design parameters to achieve those clinical benefits, much less is known regarding the design of nanoDDS capable of enhancing tumor penetration of DOX (and other drugs), which is another major factor leading to DOX's reduced efficacy. The purpose of this study was to design a dendrimer-based nanoDDS capable of enhancing the penetration of DOX as measured in an in vitro 3D lung tumor model and to correlate those results with its efficacy. Spheroids formed with the A549 human lung adenocarcinoma cells/murine fibroblast cell line (NIH/3T3 cell line) are shown to produce the essential components of the extracellular matrix (ECM), which is known as a physical barrier that hinders the transport of DOX. DOX was conjugated to generation 4 succinamic acid-terminated poly(amido-amine) (PAMAM) dendrimers (G4SA) through an enzyme-liable tetrapeptide (G4SA-GFLG-DOX), resulting in a nanoDDS with ∼5.5 DOX, -17 mV surface (ζ) potential, and a 10 nm hydrodynamic diameter (HD). The penetration of DOX to the core of the spheroid in terms of DOX fluorescence was determined to be 3.1-fold greater compared to free DOX, which positively correlated with enhanced efficacy as measured by the Caspase 3/7 assay. This improved penetration happens as the interactions between the G4SA-GFLG-DOX and the highly negatively charged ECM are minimized by shielding the protonatable amine of DOX upon conjugation, and the HD of the conjugate is kept smaller than the estimated mesh size of the ECM. Interestingly, the conjugate provided more specificity for DOX to tumor cells compared to fibroblasts, while free DOX is equally distributed in both tumor and fibroblasts as assessed in the coculture spheroids. Growth inhibition studies show that the released DOX maintains its activity and leads to tumor reduction to the same extent as free DOX. The results obtained here are of relevance for the design of dendrimer-based nanoDDS and for the treatment of solid tumors as they provide critical information regarding desirable surface characteristics and sizes for efficient tumor penetration.

Local Targeting of Lung-Tumor-Associated Macrophages with Pulmonary Delivery of a CSF-1R Inhibitor for the Treatment of Breast Cancer Lung Metastases.

The lungs are major sites of metastases for several cancer types, including breast cancer (BC). Prognosis and quality of life of BC patients that develop pulmonary metastases are negatively impacted. The development of strategies to slow the growth and relieve the symptoms of BC lung metastases (BCLM) is thus an important goal in the management of BC. However, systemically administered first line small molecule chemotherapeutics have poor pharmacokinetic profiles and biodistribution to the lungs and significant off-target toxicity, severely compromising their effectiveness. In this work, we propose the local delivery of add-on immunotherapy to the lungs to support first line chemotherapy treatment of advanced BC. In a syngeneic murine model of BCLM, we show that local pulmonary administration (p.a.) of PLX-3397 (PLX), a colony-stimulating factor 1 receptor inhibitor (CSF-1Ri), is capable of overcoming physiological barriers of the lung epithelium, penetrating the tumor microenvironment (TME), and decreasing phosphorylation of CSF-1 receptors, as shown by the Western blot of lung tumor nodules. That inhibition is accompanied by an overall decrease in the abundance of protumorigenic (M2-like) macrophages in the TME, with a concomitant increase in the amount of antitumor (M1-like) macrophages when compared to the vehicle-treated control. These effects with PLX (p.a.) were achieved using a much smaller dose (1 mg/kg, every other day) compared to the systemic doses typically used in preclinical studies (40-800 mg/kg/day). As an additive in combination with intravenous (i.v.) administration of paclitaxel (PTX), PLX (p.a.) leads to a decrease in tumor burden without additional toxicity. These results suggested that the proposed immunochemotherapy, with regional pulmonary delivery of PLX along with the i.v. standard of care chemotherapy, may lead to new opportunities to improve treatment, quality of life, and survival of patients with BCLM.

Nanoparticles of pH-Responsive, PEG-Doxorubicin Conjugates: Interaction with an in Vitro Model of Lung Adenocarcinoma and Their Direct Formulation in Propellant-Based Portable Inhalers.

Pulmonary administration of polymer drug conjugates is of great potential clinical significance for treating lung cancer as such regimen significantly increases local drug concentrations while decreases systemic and local side effects. In this work, we demonstrate that nanoparticles prepared with methoxypoly(ethylene glycol) (mPEG)-doxorubicin (DOX) conjugates (mPEG-DOX) that have a pH-sensitive imine bond (Schiff base) can at the same time work as efficient carriers for DOX to kill cancer cells and also as a strategy to directly formulate nanoparticles in propellant-based inhalers. Nanoparticles prepared by precipitation in water had a diameter in the range between 100 and 120 nm. We investigated the effects of molecular weight (MW) of mPEG (1K, 2K, and 5K Da) on the in vitro release kinetics, cellular internalization, and cytotoxicity on in vitro model of lung adenocarcinoma and aerosol characters. It is observed that the DOX released from mPEG-DOX nanoparticles was significantly accelerated in acidic environment, pH 5.5 (endosomal/lysosomal pH) in comparison with pH 7.4 (physiological pH), as designed. Release of DOX from mPEG1K-DOX nanoparticles was significantly greater than those from mPEG2K and mPEG5K counterparts. In vitro cytotoxicity of nanoparticles followed the sequence of mPEG1K-DOX > free DOX > mPEG2K-DOX ≫ mPEG5K-DOX, a trend closely following their rate and extent of cellular internalization. mPEG-DOX nanoparticles with mPEG1K and mPEG2K were directly dispersed in hydrofluoroalkane (HFA), while a trace of ethanol was required to disperse mPEG5K-DOX nanoparticles in HFA. These pMDI formulations with high physical stability in HFAs display superior aerosol characteristics conducive to deep lung deposition. The fine particle fractions of these formulations ranged from 40-60%, higher than those of commercial products. Such formulations prepared from nanoparticles of pH-sensitive PEG-drug conjugates may also be envisioned to be extended to formulate other hydrophobic drugs for local delivery with propellant-based inhalers to other pulmonary disorders, thus broadening the impact of the proposed strategy.

Conjugation to Poly(amidoamine) Dendrimers and Pulmonary Delivery Reduce Cardiac Accumulation and Enhance Antitumor Activity of Doxorubicin in Lung Metastasis.

Lung is one of the most common sites to which almost all other primary tumors metastasize. The major challenges in the chemotherapy of lung metastases include the low drug concentration found in the tumors and high systemic toxicity upon systemic administration. In this study, we combine local lung delivery and the use of nanocarrier-based systems for improving pharmacokinetics and biodistribution of the therapeutics to fight lung metastases. We investigate the impact of the conjugation of doxorubicin (DOX) to carboxyl-terminated poly(amidoamine) dendrimers (PAMAM) through a bond that allows for intracellular-triggered release, and the effect of pulmonary delivery of the dendrimer-DOX conjugate in decreasing tumor burden in a lung metastasis model. The results show a dramatic increase in efficacy of DOX treatment of the melanoma (B16-F10) lung metastasis mouse model upon pulmonary administration of the drug, as indicated by decreased tumor burden (lung weight) and increased survival rates of the animals (male C57BL/6) when compared to iv delivery. Conjugation of DOX further increased the therapeutic efficacy upon lung delivery as indicated by the smaller number of nodules observed in the lungs when compared to free DOX. These results are in agreement with the biodistribution characteristics of the DOX upon pulmonary delivery, which showed a longer lung accumulation/retention compared to iv administration. The distribution of DOX to the heart tissue is also significantly decreased upon pulmonary administration, and further decreased upon conjugation. The results show, therefore, that pulmonary administration of DOX combined to conjugation to PAMAM dendrimer through an intracellular labile bond is a potential strategy to enhance the therapeutic efficacy and decrease systemic toxicity of DOX.

Effect of the Conjugation Density of Triphenylphosphonium Cation on the Mitochondrial Targeting of Poly(amidoamine) Dendrimers.

Many clinically relevant diseases with known poor therapeutic outcomes, including cancer and neurodegenerative disorders, have been directly linked to mitochondrial dysfunction. The ability to efficiently target therapeutics to intracellular organelles such as mitochondria may represent new opportunities for the effective treatment of such ailments. The present study reports the synthesis, cellular uptake, cytotoxicity, and mitochondrial colocalization of conjugates of triphenylphosphonium cation (TPP) to amine-terminated, generation 4, poly(amidoamine) (PAMAM) dendrimer (G4NH2) nanocarriers. The mitochondrial-targeting moiety TPP was either directly conjugated to G4NH2 (G4NH2-TPP) or to the dendrimer through a flexible polyethylene glycol (PEG) linker (G4NH2-PEGTPP). Conjugation was done at various TPP densities to assess their biological activity and potential for mitochondrial-targeted drug delivery. Tests in an in vitro model of the human alveolar carcinoma (A549 cells) showed that even at a low TPP density (∼5 TPP) both the cellular internalization and mitochondrial targeting increase significantly, as determined by fluorescence activated cell sorting (FACS) and confocal microscopy (CM), respectively. At a density of ∼10 TPP per G4NH2, further increase in cellular internalization and mitochondrial targeting was achieved. However, at this higher density, the nanocarriers also showed pronounced cytotoxicity. It was observed that the toxicity of the conjugates is decreased upon the addition of a PEG linker between the dendrimer and TPP (G4NH2-PEGTPP), while the mitochondrial targeting ability of the nanocarriers is not affected as the PEG density increases. The proposed strategies indicate that TPP-conjugated G4NH2 dendrimers represent a potentially viable strategy for the targeting of therapeutic molecules to mitochondria, which may help improve therapeutic outcomes of diseases related to mitochondrial dysfunction.

Megalin-targeting liposomes for placental drug delivery.

Every year, complications during pregnancy affect more than 26 million women. Some of those diseases are associated with significant morbidity and mortality, as is the case of preeclampsia, the main cause of maternal deaths globally. The ability to improve the delivery of drugs to the placenta upon administration to the mother may offer new opportunities in the treatment of diseases of pregnancy. The objective of this study was to develop megalin-targeting liposome nanocarriers for placental drug delivery. Megalin is a transmembrane protein involved in clathrin-mediated endocytic processes, and is expressed in the syncytiotrophoblast (SynT), an epithelial layer at maternal-fetal interface. Targeting megalin thus offers an opportunity for the liposomes to hitchhike into the SynT, thus enriching the concentration of any associated therapeutic cargo in the placental tissue. PEGylated (2 KDa) lipids were modified with gentamicin (GM), a substrate to megalin receptors as we have shown in earlier studies, and used to prepare placental-targeting liposomes. The ability of the targeting liposomes to enhance accumulation of a fluorescence probe was assessed in an in vivo placental model - timed-pregnant Balb/c mice at gestational day (GD) 18.5. The targeting liposomes containing 10 mol% GM-modified lipids increased the accumulation of the conjugated fluorescence probe in the placenta with a total accumulation of 2.8% of the initial dose, which corresponds to a 94 fold increase in accumulation compared to the free probe (p < .0001), and 2-4 fold accumulation compared to the non-targeting control liposomes (p < .0001), as measured by both tissue extraction assay and ex vivo imaging. Furthermore, confocal images of placental SynT cross-sections show a 3-fold increase of the targeting liposomes compared with the non-targeting liposomes. The rate and extent of uptake of a fluorescent probe encapsulated within targeting liposomes was also probed in an in vitro model of the human placental barrier (polarized BeWo monolayers) using flow cytometry. Targeting liposomes containing 5 mol% GM-modified lipids enhanced the uptake of the probe by 1.5 fold compared to the non-targeting control. An increase to 10 mol% of the modified lipid resulted in further enhancement in uptake, which was 2 fold greater compared to control. In a competition assay, inhibition of the megalin receptors resulted in a significant reduction in uptake of the fluorescence probe encapsulated in GM-modified liposomes compared to the uptake without free inhibitor (p < .0001), implicating the involvement of megalin receptor in the internalization of the liposomes. Taken together, these results demonstrate that megalin-targeted liposomes may offer an opportunity to enhance the delivery of therapeutics to the placenta for the treatment of diseases of pregnancy.