Qian Zhong

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.

Poly(amidoamine) Dendrimer–Doxorubicin Conjugates: In Vitro Characteristics and Pseudosolution Formulation in Pressurized Metered-Dose Inhalers

Lung cancers are the leading cause of cancer death for both men and women. A series of PEGylated poly(amidoamine) dendrimer-based doxorubicin (DOX) nanocarriers (G3NH2-mPEG-nDOX) were synthesized and their chemistry tailored for the development of novel pseudosolution formulations in propellant-based metered-dose inhalers (pMDIs) with enhanced aerosol characteristics. A pH-labile bond was used to conjugate DOX to dendrimer for controlled intracellular release. We employed a two-step PEGylation strategy to cover a range of DOX loading and PEGylation density. We investigated the impact of pH, PEGylation density, and DOX payload on the release of DOX from the conjugate. We also determined the cellular internalization of the conjugate, the intracellular release kinetics of DOX from the conjugate, and their ability to kill human alveolar carcinoma cells (A549). The acid-labile conjugates sustained the release of DOX in acidic medium, and also intracellularly, as determined by nuclear colocalization studies with confocal microscopy. Meanwhile, DOX was retained in the conjugate at extracellular physiological conditions, indicating their potential to achieve spatial and temporal controlled release profiles. We also observed that the kinetics of cellular entry of the conjugates with DOX increased significantly compared to free DOX. Due to controlled release, the G3NH2-mPEG-nDOX conjugates showed time-dependent cell kill, but their cell kill ability was comparable to free DOX, which suggests their potential in vivo as compared to free DOX. The conjugates were formulated in pMDIs as pseudosolution formulations, with the help of a minimum amount of cosolvent (ethanol; <0.4%; v/v). The physical stability and aerosol characteristics of the conjugates were controlled by the PEGylation density of the carriers: the higher the PEG density, the better the dispersibility and the better the deep lung deposition of the conjugates (fine particle fraction up to ca. 80%).

ERα mediates alcohol-induced deregulation of Pol III genes in breast cancer cells

The association of alcohol consumption and breast cancer is more pronounced in cases that are positive for estrogen receptor (ER+) than in cases that are negative (ER-). Its mechanism remains to be determined. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, increasing translational capacity to promote cell transformation and tumor formation. Here, we report that alcohol increases Pol III gene transcription in both normal and cancer breast cell lines. The induction in ER+ breast cancer cells (MCF-7) is significantly higher than in ER- normal breast cells (MCF-10A, MCF-10F and MCF-12A) and is correlated with ER expression. E2 causes <2-fold increase in Pol III gene transcription. The addition of ethanol to this system now produces a 10-15-fold increase. Ethanol increases ERα expression, resulting in an increase in Brf1 protein and mRNA levels. In addition, ethanol markedly stimulates phosphorylation of JNK1, but not JNK2. Inhibition of JNK1 decreases ERE-Luc reporter activity and represses expression of ERα, Brf1 and Pol III genes. Reduction of ERα by its small interfering RNA represses Brf1 and Pol III gene transcription. Ethanol with E2 produces larger and more numerous colonies. Repression of ERα or Brf1 inhibits alcohol-induced cell transformation. Together, these results support the idea that alcohol increases ERα expression through JNK1 to elevate Brf1 expression and Pol III gene transcription to bring about greater phenotypic changes. These studies demonstrate that ERα mediates Pol III gene transcription through Brf1, suggesting that ERα may play a critical role in alcohol-induced deregulation of Pol III genes in ER+ breast cancer development.

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.

Phosphorylation of histone H3 serine 28 modulates RNA polymerase III-dependent transcription

Deregulation of RNA polymerase III (Pol III) transcription enhances cellular tRNAs and 5S rRNA production, leading to an increase in translational capacity to promote cell proliferation, transformation and tumor formation. Phosphorylation of histone H3 (H3ph) is induced by tumor promoters (EGF, UV and TPA) and immediate early genes, such as c-myc, c-jun and c-fos. However, it remains to be determined whether H3ph is involved in RNA Pol III transcription. Here, we report that EGF strongly induced H3ph at serine 28 (H3S28ph). EGF significantly increased transcription of RNA Pol III-dependent genes (Pol III genes), tRNALeu, tRNATyr, 5S rRNA and 7SL RNA. Inhibition of EGFR, but not PI3K, reduced both H3S28ph and tRNALeu and 5S rRNA transcription. EGF enhanced occupancy of H3S28ph in the promoters of tRNALeu and 5S rRNA. Further analysis indicates that EGF augmented cellular levels of protein and mRNA of TFIIIB subunits, Brf1 and TATA box-binding protein (TBP). Brf1 is a specific transcription factor for RNA Pol III genes. EGF enhanced occupancy of H3S28ph in the Brf1 and TBP promoters. Inhibition of H3S28ph by mutant H3S28A repressed Brf1, TBP and tRNALeu and 5S rRNA expression and decreased occupancy of H3S28ph in their promoters. Reduction of Brf1 significantly decreased tRNALeu and 5S rRNA transcription and repressed EGF-induced anchorage-independent growth. Blocking H3S28ph signaling by using mutant H3S28A reduced EGF-induced cell transformation. Together, these results indicate that EGF activates EGFR signaling to induce H3S28ph, which, in turn, upregulates tRNALeu and 5S rRNA transcription through Brf1 and TBP and promotes cell transformation. The studies demonstrate that epigenetic modification of H3S28ph has a critical role in the activity of Pol III genes.

Role of phosphorylated histone H3 serine 10 in DEN-induced deregulation of Pol III genes and cell proliferation and transformation

The products of Pol III genes (RNA polymerase III-dependent genes), such as tRNAs and 5S rRNA, are elevated in both transformed and tumor cells suggesting that they play a crucial role in tumorigenesis. An increase in Brf1 (TFIIIB-related factor 1), a subunit of TFIIIB, augments Pol III gene transcription and is sufficient for cell transformation and tumor formation. We have demonstrated that enhancement of Brf1 and Pol III gene expression is associated with the occurrences of hepatocellular carcinoma (HCC) in mice. This suggests that Brf1 may be a key molecule during HCC development. Diethylnitrosamine (DEN), a chemical carcinogen, has been used to induce HCC in rodents. To determine the role of Brf1 and the epigenetic-regulating events in cell proliferation and transformation, hepatocytes were treated with DEN. The results indicate that DEN increases proliferation and transformation of AML-12 cells. DEN enhanced Brf1 expression and tRNA(Leu) and 5S rRNA transcription, as well as H3S10ph (phosphorylation of histone H3 serine 10). Interestingly, DEN-induced Pol III gene transcription and H3S10ph in tumor cells of liver are significantly higher than in non-tumor cells. Inhibition of H3S10ph by H3S10A attenuates the induction of Brf1 and Pol III genes. Further analysis indicates that H3S10ph occupies the promoters of Brf1 and Pol III genes to modulate their expression. Blocking H3S10ph represses cell proliferation and transformation. These results demonstrate that DEN induces H3S10ph, which mediate Brf1 expression, including but not limited Brf1-dependent genes, to upregulate Pol III gene transcription, resulting in an increase in cell proliferation and transformation.

TPP-dendrimer nanocarriers for siRNA delivery to the pulmonary epithelium and their dry powder and metered-dose inhaler formulations

The regulation of genes utilizing the RNA interference (RNAi) mechanism via the delivery of synthetic siRNA has great potential in the treatment of a variety of lung diseases. However, the delivery of siRNA to the lungs is challenging due to the poor bioavailability of siRNA when delivered intraveneously, and difficulty in formulating and maintaining the activity of free siRNA when delivered directly to the lungs using inhalation devices. The use of non-viral vectors such as cationic dendrimers can help enhance the stability of siRNA and its delivery to the cell cytosol. Therefore, in this work, we investigate the ability of a triphenylphosphonium (TPP) modified generation 4 poly(amidoamine) (PAMAM) dendrimer (G4NH2-TPP) to enhance the in vitro transfection efficiency of siRNA in a model of the pulmonary epithelium and their aerosol formulations in pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs). Complexes of siRNA and G4NH2-TPP were prepared with varying TPP densities and increasing N/P ratios. The complexation efficiency was modulated by the presence of the TPP on the dendrimer surface, allowing for a looser complexation compared to unmodified dendrimer as determined by gel electrophoresis and polyanion competition assay. An increase in TPP density and N/P ratio led to an increase in the in vitro gene knockdown of stably green fluorescent protein (eGFP) expressing lung alveolar epithelial (A549) cells. G4NH2-12TPP dendriplexes (G4NH2 PAMAM dendrimers containing 12 TPP molecules on the surface complexed with siRNA) at N/P ratio 30 showed the highest in vitro gene knockdown efficiency. To assess the potential of TPP-dendriplexes for pulmonary use, we also developed micron particle technologies for both pMDIs and DPIs and determined their aerosol characteristics utilizing an Andersen Cascade Impactor (ACI). Mannitol microparticles encapsulating 12TPP-dendriplexes were shown to be effective in producing aerosols suitable for deep lung deposition for both pMDI formulations (fine particle fraction of 50-53%) and DPI formulations (fine particle fraction of 39%) with no impact on the in vitro gene knockdown efficiency of the siRNA. This work demonstrates the potential benefits of utilizing TPP-conjugated dendrimers in the formation of dendriplexes for siRNA delivery to the pulmonary epithelium and their aerosol formulation for local delivery to the lungs using portable inhalers.

Dendrimer-conjugated peptide vaccine enhances clearance of Chlamydia trachomatis genital infection

Peptide-based vaccines have emerged in recent years as promising candidates in the prevention of infectious diseases. However, there are many challenges to maintaining in vivo peptide stability and enhancement of peptide immunogenicity to generate protective immunity which enhances clearance of infections. Here, a dendrimer-based carrier system is proposed for peptide-based vaccine delivery, and shows its anti-microbial feasibility in a mouse model of Chlamydia trachomatis. Chlamydiae are the most prevalent sexually transmitted bacteria worldwide, and also the causal agent of trachoma, the leading cause of preventable infectious blindness. In spite of the prevalence of this infectious agent and the many previous vaccine-related studies, there is no vaccine commercially available. The carrier system proposed consists of generation 4, hydroxyl-terminated, polyamidoamine (PAMAM) dendrimers (G4OH), to which a peptide mimic of a chlamydial glycolipid antigen-Peptide 4 (Pep4, AFPQFRSATLLL) was conjugated through an ester bond. The ester bond between G4OH and Pep4 is expected to break down mainly in the intracellular environment for antigen presentation. Pep4 conjugated to dendrimer induced Chlamydia-specific serum antibodies after subcutaneous immunizations. Further, this new vaccine formulation significantly protected immunized animals from vaginal challenge with infectious Chlamydia trachomatis, and it reduced infectious loads and tissue (genital tract) damage. Pep4 conjugated to G4OH or only mixed with peptide provided enhanced protection compared to Pep4 and adjuvant (i.e. alum), suggesting a potential adjuvant effect of the PAMAM dendrimer. Combined, these results demonstrate that hydroxyl-terminated PAMAM dendrimer is a promising polymeric nanocarrier platform for the delivery of peptide vaccines and this approach has potential to be expanded to other infectious intracellular bacteria and viruses of public health significance.

Alteration of BRCA1 expression affects alcohol-induced transcription of RNA Pol III-dependent genes.

Emerging evidence has indicated that alcohol consumption is an established risk factor for breast cancer. Deregulation of RNA polymerase III (Pol III) transcription enhances cellular Pol III gene production, leading to an increase in translational capacity to promote cell transformation and tumor formation. We have reported that alcohol intake increases Pol III gene transcription to promote cell transformation and tumor formation in vitro and in vivo. Studies revealed that tumor suppressors, pRb, p53, PTEN and Maf1 repress the transcription of Pol III genes. BRCA1 is a tumor suppressor and its mutation is tightly related to breast cancer development. However, it is not clear whether BRCA1 expression affects alcohol-induced transcription of Pol III genes. At the present studies, we report that restoring BRCA1 in HCC 1937 cells, which is a BRCA1 deficient cell line, represses Pol III gene transcription. Expressing mutant or truncated BRCA1 in these cells does not affect the ability of repression on Pol III genes. Our analysis has demonstrated that alcohol induces Pol III gene transcription. More importantly, overexpression of BRCA1 in estrogen receptor positive (ER+) breast cancer cells (MCF-7) decreases the induction of tRNA(Leu) and 5S rRNA genes by alcohol, whereas reduction of BRCA1 by its siRNA slightly increases the transcription of the class of genes. This suggests that BRCA1 is associated with alcohol-induced deregulation of Pol III genes. These studies for the first time demonstrate the role of BRCA1 in induction of Pol III genes by alcohol and uncover a novel mechanism of alcohol-associated breast cancer.

Cellular internalization and transport of biodegradable polyester dendrimers on a model of the pulmonary epithelium and their formulation in pressurized metered-dose inhalers

The purpose of this study was to evaluate the effect of generation and surface PEGylation of degradable polyester-based dendrimers nanocarriers on their interactions with an in vitro model of the pulmonary epithelium as well as to assess the ability to formulate such carriers in propellant-based, portable oral-inhalation devices to determine their potential for local and systemic delivery of drugs to and through the lungs. Hydroxyl (-OH) terminated polyester dendrimers of generation 3 and 4 (G3, and G4) were synthesized using a divergent approach. G4 was surface-modified with PEG (1,000Da). All dendrimers and their building blocks were determined to be highly compatible with the model pulmonary epithelium, with toxicity profiles much more favorable than non-degradable polyamidoamine dendrimers (PAMAM). The transport of the species from the apical to basolateral side across polarized Calu-3 monolayers showed to be generation and surface-chemistry (PEGylation) dependent. The extent of the transport is modulated by their interaction with the polarized epithelium and their transient opening of the tight junctions. G3 was the one most efficiently internalized by the epithelium, and had a small impact on the integrity of the monolayer. On the other hand, the PEGylated G4 was the one least internalized by the polarized epithelium, and at the same time had a more pronounced transient impact on the cellular junctions, resulting in more efficient transport across the cell monolayer. PEGylation of the dendrimer surface played other roles as well. PEGylation modulated the degradation profile of the dendrimer, slowing the process in a step-wise fashion - first the PEG layer is shed and then the dendrimer starts degrading. PEGylation also helped increase the solvation of the nanocarriers by the hydrofluoroalkane propellant used in pressurized metered-dose inhalers, resulting in formulations with excellent dispersibility and aerosol quality (deep lung deposition of 88.5%), despite their very small geometric diameter. The combined in vitro and formulation performance results shown here demonstrated that degradable, modified polyester dendrimers may serve as a valuable platform that can be tailored to target the lung tissue for treating local diseases, or the circulation, using the lungs as pathway to the bloodstream.