Hien T. T. Duong

Polymerization-induced self-assembly (PISA) - Control over the morphology of nanoparticles for drug delivery applications

In this paper, we describe the synthesis of asymmetric functional POEGMA-b-P(ST-co-VBA) copolymers in methanol, yielding in one-pot polymerization a range of nanoparticle morphologies, including spherical micelles, worm-like, rod-like micelles and vesicles. The presence of the aldehyde group was then exploited to form crosslinks or to conjugate chemotherapy compounds, such as doxorubicin, via pH-breakable bonds (Schiff base or imine) directly to the preformed nanoparticles. The influence of the nanoparticle morphologies on the MCF-7 breast cancer cell line uptake was investigated using flow cytometry and confocal microscopy. Finally, the IC50 of DOX, following nanoparticle delivery, was studied showing significant influence of the nanoparticle carrier morphology on therapeutic efficacy for breast cancer.

Organo-photocatalysts for photoinduced electron transfer-reversible addition–fragmentation chain transfer (PET-RAFT) polymerization

In this article, we are investigating a series of organo-dyes, including methylene blue, fluorescein, rhodamine 6G, Nile red and eosin Y, to perform a visible light-mediated controlled/“living” radical polymerization of methacrylates. We demonstrate that eosin Y and fluorescein were efficient catalysts to activate a photoinduced electron transfer-reversible addition–fragmentation chain transfer (PET-RAFT) mechanism. The concentration of eosin Y was varied from 10 to 100 ppm with respect to monomers. This polymerization technique yielded well-defined (co)polymers with a good control of the molecular weights ranging from 10 000 to 100 000 g mol−1 and low polydispersities (PDI < 1.30). A variety of functional monomers, including N,N-dimethylaminoethyl methacrylate, hydroxyl ethyl methacrylate, pentafluorophenyl methacrylate, glycidyl methacrylate, oligoethylene glycol methyl ether methacrylate (OEGMA), and methacrylic acid, were successfully polymerized. Finally, the addition of a tertiary amine, such as triethylamine, afforded the polymerization in the presence of air via a reductive quenching cycle. Different diblock polymethacrylate copolymers, i.e. PMMA-b-POEGMA and PMMA-b-PMMA, were prepared to demonstrate the high end group fidelity.

Synthesis of Functional Core, Star Polymers via RAFT Polymerization for Drug Delivery Applications

Poly(oligoethylene glycol) methyl ether acrylate was polymerized via reversible addition fragmentation transfer polymerization (RAFT), and then chain extended in the presence of both a cross-linker and vinyl benzaldehyde (VBA), yielding monodisperse star polymers. The presence of aldehyde groups in the core was exploited to attach doxorubicin. The drug loading was controlled by the amount of VBA incorporated (until 28 wt% in drug). The doxorubicin release was studied at pH = 5.5 and 7.4; conditions representative of endosomal and extra cellular environments. In vitro studies revealed that the doxorubicin-conjugated star polymers had a level of cytotoxicity comparable to that found for free doxorubicin. Confocal microscopy and flow cytometry studies confirmed efficient cell uptake of the star polymers.

Core-Cross-Linked Micelles Synthesized by Clicking Bifunctional Pt(IV) Anticancer Drugs to Isocyanates

Most low molecular weight platinum-based anticancer drugs have a short circulation time in the bloodstream. One of the potential strategies to improve the targeted delivery of cisplatin and prolong its circulation is via the use of nanocarriers. An improved drug delivery system was developed via reversible addition-fragmentation chain transfer (RAFT) polymerization. In a one-pot reaction, the incorporation of anticancer drug and core cross-linking was simultaneously carried out by using the highly effective reaction of isocyanate groups in the core of the polymeric micelles poly(oligo(ethylene glycol) methyl ether methacrylate)-block-poly(styrene-co-3-isopropenyl-α,α-dimethylbenzyl isocyanate) (POEGMA-block-P(STY-co-TMI)) with amine groups in the prepared platinum(IV) drug. The micelles with platinum(IV) incorporated with a size of 36 nm were very stable in water. In a reductive environment, in this study simulated using ascorbate, the drug was released at a slow rate of 82% in 22 days and at the same time the cross-linked micelle broke down into free block copolymers as evidenced using inductively coupled plasma-mass spectrometer (ICP-MS), size exclusion chromatography (SEC), and dynamic light scattering (DLS). The in vitro study also revealed the promising antitumor activity of prepared platinum(IV) drugs encapsulated into the micelle structure.

Magnetic nanoparticles with diblock glycopolymer shells give lectin concentration-dependent MRI signals and selective cell uptake

Multivalent glycopolymers exhibit a strong affinity for specific lectin proteins depending on their specific carbohydrate functionality. In this work, we report a facile one-pot synthesis of diblock PEG-glycopolymers using a combination of Cu(0) mediated living radical polymerization and click chemistry to attach three different carbohydrates, α-D-mannose, α-D-glucose and β-D-glucose, to iron oxide nanoparticle (IONP) surfaces. The resultant IONP@P(OEGA)-b-P(sugar) nanoparticles were then extensively characterized using a wide range of analytical techniques, including ATR-FTIR, XPS and TEM. Interestingly, α-D-mannose functionalized IONPs, (IONP@P(OEGA)-b-P(N3Man)), exhibited high r2 transverse relaxivity when measured in a 9.4 T MRI. A significant change in T2 relaxation was observed following binding to the lectin concanavalin A (Con A), with a response proportional to the lectin concentration. The results reported herein indicate that the specific binding of lectin to nanoparticle surfaces can be quantitatively detected using MRI, showing significant promise for future diagnostic applications. Additionally we found a significant improvement in cell uptake for IONPs functionalized with α-D-mannose, in a lung cancer cell line (A549).

Functional Iron Oxide Magnetic Nanoparticles with Hyperthermia‐Induced Drug Release Ability by Using a Combination of Orthogonal Click Reactions

Click and drug: A combination of orthogonal click reactions is employed for the preparation of functional iron oxide nanoparticles (IONPs) that show unprecedented hyperthermia-induced drug release through a magnetically stimulated retro-Diels-Alder (rDA) process. Magnetic stimulation induces sufficient local energy in close proximity to the cycloadduct to initiate the rDA process

Folate Conjugation to Polymeric Micelles via Boronic Acid Ester to Deliver Platinum Drugs to Ovarian Cancer Cell Lines

In this study, a novel technique was used for the reversible attachment of folic acid on the surface of polymeric micelles for a tumor-specific drug delivery system. The reversible conjugation is based on the interaction between phenylboronic acid (PBA) and dopamine to form a borate ester. The conjugation is fast and efficient and in vitro experiments via confocal fluorescent microscopy show that the linker is stable in for several hours. Reversible addition-fragmentation chain transfer (RAFT) polymerization was used to synthesize two various sized water-soluble block copolymer of oligoethylene glycol methylether methacylate and methyl acrylic acid (POEGMEMA(35)-b-PMAA(200) and POEGMEMA(26)-b-PMAA(90)). The platinum drug, oxoplatin, was then subsequently attached to the polymer via ester formation leading to platinum loading of 12 wt % as determined by TGA. The platinum-induced amphiphilic block copolymers that consequently led to the formation of micelles of sizes 150 and 20 nm in an aqueous environment with the longer PMAA block forming larger micelles. The small micelles were in addition cross-linked using 1,8-diaminooctane to further stabilize their structure. The targeting ability of folate conjugated polymeric micelles was investigated against two types of tumor cell lines: A549 (-FR) and OVCAR-3 (+FR). The cell line growth inhibitory efficacy of material synthesized was evaluated by using SRB method. The results revealed that folate conjugated micelles showed higher activity in FR + OVCAR-3 cells but not in FR - A549 cells. Similar results were obtained for both small and large micelles without the conjugation of folate. Comparing large and small micelles it can be observed that larger micelles are more efficient, which has been attributed to the lower stability of the smaller micelles. Micelle stabilization via cross-linking could indeed increase the toxicity of the drug carrier.

Effective delivery of siRNA into cancer cells and tumors using well-defined biodegradable cationic star polymers.

Cancer is one of the most common causes of death worldwide. Two types of cancer that have high mortality rates are pancreatic and lung cancer. Despite improvements in treatment strategies, resistance to chemotherapy and the presence of metastases are common. Therefore, novel therapies which target and silence genes involved in regulating these processes are required. Short-interfering RNA (siRNA) holds great promise as a therapeutic to silence disease-causing genes. However, siRNA requires a delivery vehicle to enter the cell to allow it to silence its target gene. Herein, we report on the design and synthesis of cationic star polymers as novel delivery vehicles for siRNA to silence genes in pancreatic and lung cancer cells. Dimethylaminoethyl methacrylate (DMAEMA) was polymerized via reversible addition-fragmentation transfer polymerization (RAFT) and then chain extended in the presence of both cross-linkers N,N-bis(acryloyl)cistamine and DMAEMA, yielding biodegradable well-defined star polymers. The star polymers were characterized by transmission electron microscopy, dynamic light scattering, ζ potential, and gel permeation chromatography. Importantly, the star polymers were able to self-assemble with siRNA and form small uniform nanoparticle complexes. Moreover, the ratios of star polymer required to complex siRNA were nontoxic in both pancreatic and lung cancer cells. Treatment with star polymer-siRNA complexes resulted in uptake of siRNA into both cell lines and a significant decrease in target gene mRNA and protein levels. In addition, delivery of clinically relevant amounts of siRNA complexed to the star polymer were able to silence target gene expression by 50% in an in vivo tumor setting. Collectively, these results provide the first evidence of well-defined small cationic star polymers to deliver active siRNA to both pancreatic and lung cancer cells and may be a valuable tool to inhibit key genes involved in promoting chemotherapy drug resistance and metastases.

Dextran-Based Doxorubicin Nanocarriers with Improved Tumor Penetration

Drug delivery systems with improved tumor penetration are valuable assets as anticancer agents. A dextran-based nanocarrier system with aldehyde functionalities capable of forming an acid labile linkage with the chemotherapy drug doxorubicin was developed. Aldehyde dextran nanocarriers (ald-dex-dox) demonstrated efficacy as delivery vehicles with an IC50 of ∼300 nM against two-dimensional (2D) SK-N-BE(2) monolayers. Confocal imaging showed that the ald-dex-dox nanocarriers were rapidly internalized by SK-N-BE(2) cells. Fluorescence lifetime imaging microscopy (FLIM) analysis indicated that ald-dex-dox particles were internalized as intact complexes with the majority of the doxorubicin released from the particle four hours post uptake. Accumulation of the ald-dex-dox particles was significantly enhanced by ∼30% in the absence of glucose indicating a role for glucose and its receptors in their endocytosis. However, inhibition of clathrin dependent and independent endocytosis and macropinocytosis as well as membrane cholesterol depletion had no effect on ald-dex-dox particle accumulation. In three-dimensional (3D) SK-N-BE(2) tumor spheroids, which more closely resemble a solid tumor, the ald-dex-dox nanoparticles showed a significant improvement in efficacy over free doxorubicin, as evidenced by decreased spheroid outgrowth. Drug penetration studies in 3D demonstrated the ability of the ald-dex-dox nanocarriers to fully penetrate into a SK-N-BE(2) tumor spheroids, while doxorubicin only penetrates to a maximum distance of 50 μM. The ald-dex-dox nanocarriers represent a promising therapeutic delivery system for the treatment of solid tumors due to their unique enhanced penetration ability combined with their improved efficacy over the parent drug in 3D.

Pair correlation microscopy reveals the role of nanoparticle shape in intracellular transport and site of drug release

Nanoparticle size, surface charge and material composition are known to affect the uptake of nanoparticles by cells. However, whether nanoparticle shape affects transport across various barriers inside the cell remains unclear. Here we used pair correlation microscopy to show that polymeric nanoparticles with different shapes but identical surface chemistries moved across the various cellular barriers at different rates, ultimately defining the site of drug release. We measured how micelles, vesicles, rods and worms entered the cell and whether they escaped from the endosomal system and had access to the nucleus via the nuclear pore complex. Rods and worms, but not micelles and vesicles, entered the nucleus by passive diffusion. Improving nuclear access, for example with a nuclear localization signal, resulted in more doxorubicin release inside the nucleus and correlated with greater cytotoxicity. Our results therefore demonstrate that drug delivery across the major cellular barrier, the nuclear envelope, is important for doxorubicin efficiency and can be achieved with appropriately shaped nanoparticles.