Nucleus-Targeted, Echogenic Polymersomes for Delivering a Cancer Stemness Inhibitor to Pancreatic Cancer Cells.

Chemotherapeutic agents for treating cancers show considerable side effects, toxicity, and drug resistance. To mitigate the problems, we designed nucleus-targeted, echogenic, stimuli-responsive polymeric vesicles (polymersomes) to transport and subsequently release the encapsulated anticancer drugs within the nuclei of pancreatic cancer cells. We synthesized an alkyne-dexamethasone derivative and conjugated it to N3-polyethylene glycol (PEG)-polylactic acid (PLA) copolymer employing the Cu2+ catalyzed "Click" reaction. We prepared polymersomes from the dexamethasone-PEG-PLA conjugate along with a synthesized stimuli-responsive polymer PEG-S-S-PLA. The dexamethasone group dilates the nuclear pore complexes and transports the vesicles to the nuclei. We designed the polymersomes to release the encapsulated drugs in the presence of a high concentration of reducing agents in the nuclei of pancreatic cancer cells. We observed that the nucleus-targeted, stimuli-responsive polymersomes released 70% of encapsulated contents in the nucleus-mimicking environment in 80 min. We encapsulated the cancer stemness inhibitor BBI608 in the vesicles and observed that the BBI608 encapsulated polymersomes reduced the viability of the BxPC3 cells to 43% in three-dimensional spheroid cultures. The polymersomes were prepared following a special protocol so that they scatter ultrasound, allowing imaging by a medical ultrasound scanner. Therefore, these echogenic, targeted, stimuli-responsive, and drug-encapsulated polymersomes have the potential for trackable, targeted carrier of chemotherapeutic drugs to cancer cell nuclei.

Tissue-Penetrating, Hypoxia-Responsive Echogenic Polymersomes For Drug Delivery To Solid Tumors.

Hypoxia in solid tumors facilitates the progression of the disease, develops resistance to chemo and radiotherapy, and contributes to relapse. Due to the lack of tumor penetration, most of the reported drug carriers are unable to reach the hypoxic niches of the solid tumors. We have developed tissue-penetrating, hypoxia-responsive echogenic polymersomes to deliver anticancer drugs to solid tumors. The polymersomes are composed of a hypoxia-responsive azobenzene conjugated and a tissue penetrating peptide functionalized polylactic acid-polyethylene glycol polymer. The drug-encapsulated, hypoxia-responsive polymersomes substantially decreased the viability of pancreatic cancer cells in spheroidal cultures. Under normoxic conditions, polymersomes were echogenic at diagnostic ultrasound frequencies but lose the echogenicity under hypoxia. In-vivo imaging studies with xenograft mouse model further confirmed the ability of the polymersomes to target, penetrate, and deliver the encapsulated contents in hypoxic pancreatic tumor tissues.

Nuclear Localizing Peptide-Conjugated, Redox-Sensitive Polymersomes for Delivering Curcumin and Doxorubicin to Pancreatic Cancer Microtumors.

Improving the therapeutic index of anticancer agents is an enormous challenge. Targeting decreases the side effects of the therapeutic agents by delivering the drugs to the intended destination. Nanocarriers containing the nuclear localizing peptide sequences (NLS) translocate to the cell nuclei. However, the nuclear localization peptides are nonselective and cannot distinguish the malignant cells from the healthy counterparts. In this study, we designed a "masked" NLS peptide which is activated only in the presence of overexpressed matrix metalloproteinase-7 (MMP-7) enzyme in the pancreatic cancer microenvironment. This peptide is conjugated to the surface of redox responsive polymersomes to deliver doxorubicin and curcumin to the pancreatic cancer cell nucleus. We have tested the formulation in both two- and three-dimensional cultures of pancreatic cancer and normal cells. Our studies revealed that the drug-encapsulated polymeric vesicles are significantly more toxic toward the cancer cells (shrinking the spheroids up to 49%) compared to the normal cells (shrinking the spheroids up to 24%). This study can lead to the development of other organelle targeted drug delivery systems for various human malignancies.

Prostate-Specific Membrane Antigen Targeted Polymersomes for Delivering Mocetinostat and Docetaxel to Prostate Cancer Cell Spheroids.

Prostate cancer cells overexpress the prostate-specific membrane antigen (PSMA) receptors on the surface. Targeting the PSMA receptor creates a unique opportunity for drug delivery. Docetaxel is a Food and Drug Administration-approved drug for treating metastatic and androgen-independent prostate cancer, and mocetinostat is a potent inhibitor of class I histone deacetylases. In this study, we prepared reduction-sensitive polymersomes presenting folic acid on the surface and encapsulating either docetaxel or mocetinostat. The presence of folic acid allowed efficient targeting of the PSMA receptor and subsequent internalization of the polymeric vesicles in cultured LNCaP prostate cancer cell spheroids. The intracellular reducing agents efficiently released docetaxel and mocetinostat from the polymersomes. The combination of the two drug-encapsulated polymersome formulations significantly (p < 0.05) decreased the viability of the LNCaP cells (compared to free drugs or control) in three-dimensional spheroid cultures. The calculated combination index value indicated a synergistic effect for the combination of mocetinostat and docetaxel. Thus, our PSMA-targeted drug-encapsulated polymersomes has the potential to lead to a new direction in prostate cancer therapy that decreases the toxicity and increases the efficacy of the drug delivery systems.

Hypoxia Responsive, Tumor Penetrating Lipid Nanoparticles for Delivery of Chemotherapeutics to Pancreatic Cancer Cell Spheroids.

Solid tumors are often poorly irrigated due to structurally compromised microcirculation. Uncontrolled multiplication of cancer cells, insufficient blood flow, and the lack of enough oxygen and nutrients lead to the development of hypoxic regions in the tumor tissues. As the partial pressure of oxygen drops below the necessary level (10 psi), the cancer cells modulate their genetic makeup to survive. Hypoxia triggers tumor progression by enhancing angiogenesis, cancer stem cell production, remodeling of the extracellular matrix, and epigenetic changes in the cancer cells. However, the hypoxic regions are usually located deep in the tumors and are usually inaccessible to the intravenously injected drug carrier or the drug. Considering the designs of the reported nanoparticles, it is likely that the drug is delivered to the peripheral tumor tissues, close to the blood vessels. In this study, we prepared lipid nanoparticles (LNs) comprising the synthesized hypoxia-responsive lipid and a peptide-lipid conjugate. We observed that the resultant LNs penetrated to the hypoxic regions of the tumors. Under low oxygen partial pressure, the hypoxia-responsive lipid undergoes reduction, destabilizing the lipid membrane, and releasing encapsulated drugs from the nanoparticles. We demonstrated the results employing spheroidal cultures of the pancreatic cancer cells BxPC-3. We observed that the peptide-decorated, drug encapsulated LNs reduced the viability of pancreatic cancer cells of the spheroids to 35% under hypoxic conditions.

Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells.

Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)-azobenzene-poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors.

Sequential culture on biomimetic nanoclay scaffolds forms three-dimensional tumoroids.

In recent times, the limitation of two-dimensional cultures and complexity of in vivo models has paved the way for the development of three-dimensional models for studying cancer. Here we report the development of a new tumor model using PCL/HAPClay scaffolds seeded with a sequential culture of human mesenchymal stem cells (hMSCs) followed by human prostate cancer cells (HPCCs). This nanocomposite system is used as a test-bed for studying cancer metastasis and efficacy of anti-cancer drugs using a polymersome delivery method. A novel sequential cell culture system in three-dimensional in vitro bone model provides a unique bone mimetic environment. The hMSCs seeded scaffolds are seeded with prostate cancer cells after the hMSCs have differentiated into osteoblasts. Sequential culture on the scaffolds has shown formation of tumoroids or microtissue consisting of organized, densely packed round cells with hypoxic core regions similar to in vivo tumors. Such tumoroids are not observed on HPCC seeded scaffolds or when HPCCs sequentially cultured with human osteoblast cells. Clearly, the newly differentiated hMSCs play a vital role in the ability of cancer cells to grow into tumoroids and cause disease. The PCL/HAPclay scaffold system seeded with the sequential culture of hMSCs, and HPCCs presents a good model system for study of the interactions between prostate cancer cells and bone microenvironment. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1591-1602, 2016.

Mmp-9 responsive PEG cleavable nanovesicles for efficient delivery of chemotherapeutics to pancreatic cancer.

Significant differences in biochemical parameters between normal and tumor tissues offer an opportunity to chemically design drug carriers which respond to these changes and deliver the drugs at the desired site. For example, overexpression of the matrix metalloproteinase-9 (MMP-9) enzyme in the extracellular matrix of tumor tissues can act as a trigger to chemically modulate the drug delivery from the carriers. In this study, we have synthesized an MMP-9-cleavable, collagen mimetic lipopeptide which forms nanosized vesicles with the POPC, POPE-SS-PEG, and cholesteryl-hemisuccinate lipids. The lipopeptide retains the triple-helical conformation when incorporated into these nanovesicles. The PEG groups shield the substrate lipopeptides from hydrolysis by MMP-9. However, in the presence of elevated glutathione levels, the PEG groups are reductively removed, exposing the lipopeptides to MMP-9. The resultant peptide-bond cleavage disturbs the vesicles' lipid bilayer, leading to the release of encapsulated contents. These PEGylated nanovesicles are capable of encapsulating the anticancer drug gemcitabine with 50% efficiency. They were stable in physiological conditions and in human serum. Effective drug release was demonstrated using the pancreatic ductal carcinoma cells (PANC-1 and MIAPaCa-2) in two-dimensional and three-dimensional "tumor-like" spheroid cultures. A reduction in tumor growth was observed after intravenous administration of the gemcitabine-encapsulated nanovesicles in the xenograft model of athymic, female nude mice.

Multifunctional polymersomes for cytosolic delivery of gemcitabine and doxorubicin to cancer cells.

Although liposomes are widely used as carriers of drugs and imaging agents, they suffer from a lack of stability and the slow release of the encapsulated contents at the targeted site. Polymersomes (vesicles of amphiphilic polymers) are considerably more stable compared to liposomes; however, they also demonstrate a slow release for the encapsulated contents, limiting their efficacy as a drug-delivery tool. As a solution, we prepared and characterized echogenic polymersomes, which are programmed to release the encapsulated drugs rapidly when incubated with cytosolic concentrations of glutathione. These vesicles encapsulated air bubbles inside and efficiently reflected diagnostic-frequency ultrasound. Folate-targeted polymersomes showed an enhanced uptake by breast and pancreatic-cancer cells in a monolayer as well as in three-dimensional spheroid cultures. Polymersomes encapsulated with the anticancer drugs gemcitabine and doxorubicin showed significant cytotoxicity to these cells. With further improvements, these vesicles hold the promise to serve as multifunctional nanocarriers, offering a triggered release as well as diagnostic ultrasound imaging.

Hexanoic acid and polyethylene glycol double grafted amphiphilic chitosan for enhanced gene delivery: influence of hydrophobic and hydrophilic substitution degree.

Gene therapy holds immense potential as a future therapeutic strategy for the treatment of numerous genetic diseases which are incurable to date. Nevertheless, safe and efficient gene delivery remains the most challenging aspects of gene therapy. To overcome this difficulty a series of hexanoic acid (HA) and monomethoxy poly(ethylene glycol) (mPEG) double grafted chitosan-based (HPC) nanomicelles were developed as nonviral gene carrier. HPC polymers with various HA and mPEG substitution degrees were synthesized, and their chemical structures were confirmed by (1)H NMR spectroscopy. HPC nanomicelles exhibited excellent blood compatibility and cell viability, as demonstrated by in vitro hemolysis and MTT assay, respectively. The cationic HPC nanomicelles retained the plasmid DNA (pDNA) binding capacity of chitosan and formed stable HPC/pDNA polyplexes with diameters below 200 nm. Both hydrophobic and hydrophilic substitution resulted in suppressed nonspecific protein adsorption on HPC/pDNA polyplexes and increased pDNA dissociation. However, resistance against DNase I degradation was enhanced by HA conjugation while being inhibited by mPEG substitution. Amphiphilic modification resulted in 3-4.5-fold higher cellular uptake in human embryonic kidney 293 cells (HEK 293) mainly through clathrin-mediated pathway. The optimal HPC/pDNA polyplexes displayed 50-fold and 1.2-fold higher gene transfection compared to unmodified chitosan and Fugene, respectively, in HEK 293 cells. Moreover, both the cellular uptake and in vitro transfection study suggested a clear dependence of gene expression on the extent of HA and mPEG substitution. These findings demonstrate that amphiphilic HPC nanomicelles with the proper combination of HA and mPEG substitution could be used as a promising gene carrier for efficient gene therapy.

Polymer-coated echogenic lipid nanoparticles with dual release triggers.

Although lipid nanoparticles are promising drug delivery vehicles, passive release of encapsulated contents at the target site is often slow. Herein, we report contents release from targeted, polymer-coated, echogenic lipid nanoparticles in the cell cytoplasm by redox trigger and simultaneously enhanced by diagnostic frequency ultrasound. The lipid nanoparticles were polymerized on the external leaflet using a disulfide cross-linker. In the presence of cytosolic concentrations of glutathione, the lipid nanoparticles released 76% of encapsulated contents. Plasma concentrations of glutathione failed to release the encapsulated contents. Application of 3 MHz ultrasound for 2 min simultaneously with the reducing agent enhanced the release to 96%. Folic acid conjugated, doxorubicin-loaded nanoparticles showed enhanced uptake and higher cytotoxicity in cancer cells overexpressing the folate receptor (compared to the control). With further developments, these lipid nanoparticles have the potential to be used as multimodal nanocarriers for simultaneous targeted drug delivery and ultrasound imaging.

Glycosaminoglycan-mediated selective changes in the aggregation states, zeta potentials, and intrinsic stability of liposomes.

Though the aggregation of glycosaminoglycans (GAGs) in the presence of liposomes and divalent cations has been previously reported, the effects of different GAG species and minor changes in GAG composition on the aggregates that are formed are yet unknown. If minor changes in GAG composition produce observable changes in the liposome aggregate diameter or zeta potential, such a phenomenon may be used to detect potentially dangerous oversulfated contaminants in heparin. We studied the mechanism of the interactions between heparin and its oversulfated glycosaminoglycan contaminants with liposomes. Herein, we demonstrate that Mg(2+) acts to shield the incoming glycosaminoglycans from the negatively charged phosphate groups of the phospholipids and that changes in the aggregate diameter and zeta potential are a function of the glycosaminoglycan species and concentration as well as the liposome bilayer composition. These observations are supported by TEM studies. We have shown that the organizational states of the liposome bilayers are influenced by the presence of GAG and excess Mg(2+), resulting in a stabilizing effect that increases the T(m) value of DSPC liposomes; the magnitude of this effect is also dependent on the GAG species and concentration present. There is an inverse relationship between the percent change in aggregate diameter and the percent change in aggregate zeta potential as a function of GAG concentration in solution. Finally, we demonstrate that the diameter and zeta potential changes in POPC liposome aggregates in the presence of different oversulfated heparin contaminants at low concentrations allow for an accurate detection of oversulfated chondroitin sulfate at concentrations of as low as 1 mol %.

Fluorescent polymer-based post-translational differentiation and subtyping of breast cancer cells.

Herein, we report the application of synthesized fluorescent, water soluble polymers for post-translational subtyping and differentiation of breast cancer cells in vitro. The fluorescence emission spectra from these polymers were modulated differently in the presence of conditioned cell culture media from various breast cancer cells. These polymers differentiate at a post-translation level possibly due to their ability to interact with extracellular enzymes that are over-expressed in cancerous conditions.

Linker-induced anomalous emission of organic-molecule conjugated metal-oxide nanoparticles.

Semiconductor nanoparticles conjugated with organic- and dye-molecules to yield high efficiency visible photoluminescence (PL) hold great potential for many future technological applications. We show that folic acid (FA)-conjugated to nanosize TiO(2) and CeO(2) particles demonstrates a dramatic increase of photoemission intensity at wavelengths between 500 and 700 nm when derivatized using aminopropyl trimethoxysilane (APTMS) as spacer-linker molecules between the metal oxide and FA. Using density-functional theory (DFT) and time-dependent DFT calculations we demonstrate that the strong increase of the PL can be explained by electronic transitions between the titania surface oxygen vacancy (OV) states and the low-energy excited states of the FA/APTMS molecule anchored onto the surface oxygen bridge sites in close proximity to the OVs. We suggest this scenario to be a universal feature for a wide class of metal oxide nanoparticles, including nanoceria, possessing a similar band gap (∼3 eV) and with a large surface-vacancy-related density of electronic states. We demonstrate that the molecule-nanoparticle linker can play a crucial role in tuning the electronic and optical properties of nanosystems by bringing optically active parts of the molecule and of the surface close to each other.

Differentiation of prostate cancer cells using flexible fluorescent polymers.

Using water-soluble, fluorescent, flexible polymers, we have devised a novel methodology for identification and differentiation of prostate cancer cells. Using a stepwise linear discriminant analysis, we demonstrate that the differential modulations of the polymer emission intensities in the presence of conditioned cell culture media can be used to distinguish between prostate cancer subtypes and between cancerous and noncancer cells. The differences in the compositions of the conditioned cell culture media are likely contributing to different fluorescence spectral patterns of the polymers. This in vitro approach may provide a novel platform for the development of an alternative prostate cancer diagnostic and subtyping technique.

Fluorescent liposomes for differential interactions with glycosaminoglycans.

We have successfully synthesized a lipid containing the pyranine dye as the hydrophilic headgroup. This lipid was incorporated into liposomes with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine as the major component. The resultant liposomes displayed differential modulations in fluorescence emission intensity in the presence of nanomolar concentrations of different glycosaminoglycans. Linear discriminant analysis of the fluorescence response data demonstrate that the liposomes are able to distinguish between different GAGs. In addition, we also demonstrate that the liposomes incorporating the pyranine lipid are able to distinguish between dilute serum from healthy individuals and serum containing elevated chondroitin sulfate (simulated serum from an Alzheimer's disease patient).

Fluorescent water soluble polymers for isozyme-selective interactions with matrix metalloproteinase-9.

Matrix metalloproteinases (MMPs) are overexpressed in various pathological conditions, including cancers. Although these isozymes have similar active sites, the patterns of exposed amino acids on their surfaces are different. Herein, we report the synthesis and molecular interactions of two water soluble, fluorescent polymers which demonstrate selective interactions with MMP-9 compared to MMP-7 and -10.

Solvent-assisted slow conversion of a dithiazole derivative produces a competitive inhibitor of peptide deformylase.

Due to its potential as an antibiotic target, E. coli peptide deformylase (PDF(Ec)) serves as a model enzyme system for inhibitor design. While investigating the structural-functional and inhibitory features of this enzyme, we unexpectedly discovered that 2-amino-5-mercapto-1,3,4-thiadiazole (AMT) served as a slow-binding inhibitor of PDF(Ec) when the above compound was dissolved only in dimethylformamide (DMF), but not in any other solvent, and allowed to age. The time dependent inhibitory potency of the DMF-dissolved AMT was correlated with the broadening of the inhibitor's 295 nm spectral band toward the visible region, concomitant with the increase in the mass of the parent compound by about 2-fold. These data led to the suggestion that DMF facilitated the slow dimerization of AMT (via the formation of a disulfide bond), and that the dimeric form of AMT served as an inhibitor for PDF(Ec). The latter is not caused by the simple oxidation of sulfhydryl groups by oxidizing agents such as H(2)O(2). Newly synthesized dimeric/dithiolated form of AMT ("bis-AMT") exhibited similar spectral and inhibitory features as given by the parent compound when incubated with DMF. The computer graphic modeling data revealed that bis-AMT could be reliably accommodated within the active site pocket of PDF(Ec), and the above enzyme-ligand interaction involves coordination with the enzyme resident Ni(2+) cofactor. The mechanism of the DMF-assisted activation of AMT (generating bis-AMT), the overall microscopic pathway for the slow-binding inhibition of PDF(Ec) by bis-AMT, and the potential of bis-AMT to serve as a new class of antibiotic agent are presented.

Novel bis-(arylsulfonamide) hydroxamate-based selective MMP inhibitors.

A series of bis-(arylsulfonamide) hydroxamate inhibitors were synthesized. These compounds exhibit good potency against MMP-7 and MMP-9 depending on the nature, steric bulk, and substitution pattern of the substituents in the benzene ring. In general, the preliminary structure-activity relationships (SAR) suggest that among the DAPA hydroxamates (i) electron-rich benzene rings of the sulfonamides may produce better inhibitors than electron-poor analogs. However, potential H-bond acceptors can reverse the trend depending on the isozyme; (ii) isozyme selectivity between MMP-7 and -9 can be conferred through steric bulk and substitution pattern of the substituents in the benzene ring, and (iii) the MMP-10 inhibition pattern of the compounds paralleled that for MMP-9.

Synthesis of barbiturate-based methionine aminopeptidase-1 inhibitors.

The syntheses of a new class of barbiturate-based inhibitors for human and Escherichia Coli methionine aminopeptidase-1 (MetAP-1) are described. Some of the synthesized inhibitors show selective inhibition of the human enzyme with high potency.