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Binding Characterization of Aptamer-Drug Layered Microformulations and In Vitro Release Assessment

Tan KX, Danquah MK, Pan S, Yon LS

J Pharm Sci, 2019 09;108(9):2934-2941.
PMID: 31002808 DOI: 10.1016/j.xphs.2019.03.037

Efficient delivery of adequate active ingredients to targeted malignant cells is critical, attributing to recurrent biophysical and biochemical challenges associated with conventional pharmaceutical delivery systems. These challenges include drug leakage, low targeting capability, high systemic cytotoxicity, and poor pharmacokinetics and pharmacodynamics. Targeted delivery system is a promising development to deliver sufficient amounts of drug molecules to target cells in a controlled release pattern mode. Aptameric ligands possess unique affinity targeting capabilities which can be exploited in the design of high pay-load drug formulations to navigate active molecules to the malignant sites. This study focuses on the development of a copolymeric and multifunctional drug-loaded aptamer-conjugated poly(lactide-co-glycolic acid)-polyethylenimine (PLGA-PEI) (DPAP) delivery system, via a layer-by-layer synthesis method, using a water-in-oil-in-water double emulsion approach. The binding characteristics, targeting capability, biophysical properties, encapsulation efficiency, and drug release profile of the DPAP system were investigated under varying conditions of ionic strength, polymer composition and molecular weight (MW), and degree of PEGylation of the synthetic core. Experimental results showed increased drug release rate with increasing buffer ionic strength. DPAP particulate system obtained the highest drug release of 50% at day 9 at 1 M NaCl ionic strength. DPAP formulation, using PLGA 65:35 and PEI MW of ∼800 Da, demonstrated an encapsulation efficiency of 78.93%, and a loading capacity of 0.1605 mg bovine serum albumin per mg PLGA. DPAP (PLGA 65:35, PEI MW∼25 kDa) formulation showed a high release rate with a biphasic release profile. Experimental data depicted a lower targeting power and reduced drug release rate for the PEGylated DPAP formulations. The outcomes from the present study lay the foundation to optimize the performance of DPAP system as an effective synthetic drug carrier for targeted delivery.
Aptamer-Mediated Polymeric Vehicles for Enhanced Cell-Targeted Drug Delivery

Tan KX, Danquah MK, Sidhu A, Yon LS, Ongkudon CM

Curr Drug Targets, 2018 02 08;19(3):248-258.
PMID: 27321771 DOI: 10.2174/1389450117666160617120926

BACKGROUND: The search for smart delivery systems for enhanced pre-clinical and clinical pharmaceutical delivery and cell targeting continues to be a major biomedical research endeavor owing to differences in the physicochemical characteristics and physiological effects of drug molecules, and this affects the delivery mechanisms to elicit maximum therapeutic effects. Targeted drug delivery is a smart evolution essential to address major challenges associated with conventional drug delivery systems. These challenges mostly result in poor pharmacokinetics due to the inability of the active pharmaceutical ingredients to specifically act on malignant cells thus, causing poor therapeutic index and toxicity to surrounding normal cells. Aptamers are oligonucleotides with engineered affinities to bind specifically to their cognate targets. Aptamers have gained significant interests as effective targeting elements for enhanced therapeutic delivery as they can be generated to specifically bind to wide range of targets including proteins, peptides, ions, cells and tissues. Notwithstanding, effective delivery of aptamers as therapeutic vehicles is challenged by cell membrane electrostatic repulsion, endonuclease degradation, low pH cleavage, and binding conformation stability.
OBJECTIVE: The application of molecularly engineered biodegradable and biocompatible polymeric particles with tunable features such as surface area and chemistry, particulate size distribution and toxicity creates opportunities to develop smart aptamer-mediated delivery systems for controlled drug release.
RESULTS: This article discusses opportunities for particulate aptamer-drug formulations to advance current drug delivery modalities by navigating active ingredients through cellular and biomolecular traffic to target sites for sustained and controlled release at effective therapeutic dosages while minimizing systemic cytotoxic effects.
CONCLUSION: A proposal for a novel drug-polymer-aptamer-polymer (DPAP) design of aptamer-drug formulation with stage-wise delivery mechanism is presented to illustrate the potential efficacy of aptamer- polymer cargos for enhanced cell targeting and drug delivery.
SELEX Modifications and Bioanalytical Techniques for Aptamer-Target Binding Characterization

Tan SY, Acquah C, Sidhu A, Ongkudon CM, Yon LS, Danquah MK

Crit Rev Anal Chem, 2016 Nov;46(6):521-37.
PMID: 26980177 DOI: 10.1080/10408347.2016.1157014

The quest to improve the detection of biomolecules and cells in health and life sciences has led to the discovery and characterization of various affinity bioprobes. Libraries of synthetic oligonucleotides (ssDNA/ssRNA) with randomized sequences are employed during Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to select highly specific affinity probes called aptamers. With much focus on the generation of aptamers for a variety of target molecules, conventional SELEX protocols have been modified to develop new and improved SELEX protocols yielding highly specific and stable aptamers. Various techniques have been used to analyze the binding interactions between aptamers and their cognate molecules with associated merits and limitations. This article comprehensively reviews research advancements in the generation of aptamers, analyses physicochemical conditions affecting their binding characteristics to cellular and biomolecular targets, and discusses various field applications of aptameric binding. Biophysical techniques employed in the characterization of the molecular and binding features of aptamers to their cognate targets are also discussed.
Fulltext Characterisation of aptamer-anchored poly(EDMA-co-GMA) monolith for high throughput affinity binding

Acquah C, Chan YW, Pan S, Yon LS, Ongkudon CM, Guo H, et al.

Sci Rep, 2019 10 10;9(1):14501.
PMID: 31601836 DOI: 10.1038/s41598-019-50862-1

Immobilisation of aptameric ligands on solid stationary supports for effective binding of target molecules requires understanding of the relationship between aptamer-polymer interactions and the conditions governing the mass transfer of the binding process. Herein, key process parameters affecting the molecular anchoring of a thrombin-binding aptamer (TBA) onto polymethacrylate monolith pore surface, and the binding characteristics of the resulting macroporous aptasensor were investigated. Molecular dynamics (MD) simulations of the TBA-thrombin binding indicated enhanced Guanine 4 (G4) structural stability of TBA upon interaction with thrombin in an ionic environment. Fourier-transform infrared spectroscopy and thermogravimetric analyses were used to characterise the available functional groups and thermo-molecular stability of the immobilised polymer generated with Schiff-base activation and immobilisation scheme. The initial degradation temperature of the polymethacrylate stationary support increased with each step of the Schiff-base process: poly(Ethylene glycol Dimethacrylate-co-Glycidyl methacrylate) or poly(EDMA-co-GMA) [196.0 °C (±1.8)]; poly(EDMA-co-GMA)-Ethylenediamine [235.9 °C (±6.1)]; poly(EDMA-co-GMA)-Ethylenediamine-Glutaraldehyde [255.4 °C (±2.7)]; and aptamer-modified monolith [273.7 °C (±2.5)]. These initial temperature increments reflected in the associated endothermic energies were determined with differential scanning calorimetry. The aptameric ligand density obtained after immobilisation was 480 pmol/μL. Increase in pH and ionic concentration affected the surface charge distribution and the binding characteristics of the aptamer-modified disk-monoliths, resulting in the optimum binding pH and ionic concentration of 8.0 and 5 mM Mg2+, respectively. These results are critical in understanding and setting parametric constraints indispensable to develop and enhance the performance of aptasensors.
Fulltext Immobilization of Peroxidase on Functionalized MWCNTs-Buckypaper/Polyvinyl alcohol Nanocomposite Membrane

Jun LY, Mubarak NM, Yon LS, Bing CH, Khalid M, Jagadish P, et al.

Sci Rep, 2019 02 18;9(1):2215.
PMID: 30778111 DOI: 10.1038/s41598-019-39621-4

Surface modified Multi-walled carbon nanotubes (MWCNTs) Buckypaper/Polyvinyl Alcohol (BP/PVA) composite membrane was synthesized and utilized as support material for immobilization of Jicama peroxidase (JP). JP was successfully immobilized on the BP/PVA membrane via covalent bonding by using glutaraldehyde. The immobilization efficiency was optimized using response surface methodology (RSM) with the face-centered central composite design (FCCCD) model. The optimum enzyme immobilization efficiency was achieved at pH 6, with initial enzyme loading of 0.13 U/mL and immobilization time of 130 min. The results of BP/PVA membrane showed excellent performance in immobilization of JP with high enzyme loading of 217 mg/g and immobilization efficiency of 81.74%. The immobilized system exhibited significantly improved operational stability under various parameters, such as pH, temperature, thermal and storage stabilities when compared with free enzyme. The effective binding of peroxidase on the surface of the BP/PVA membrane was evaluated and confirmed by Field emission scanning electron microscopy (FESEM) coupled with Energy Dispersive X-Ray Spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and Thermogravimetric Analysis (TGA). This work reports the characterization results and performances of the surface modified BP/PVA membrane for peroxidase immobilization. The superior properties of JP-immobilized BP/PVA membrane make it promising new-generation nanomaterials for industrial applications.
Modelling of methylene blue adsorption using peroxidase immobilized functionalized Buckypaper/polyvinyl alcohol membrane via ant colony optimization

Jun LY, Karri RR, Mubarak NM, Yon LS, Bing CH, Khalid M, et al.

Environ Pollut, 2020 Apr;259:113940.
PMID: 31931415 DOI: 10.1016/j.envpol.2020.113940

Jicama peroxidase (JP) was covalently immobilized onto functionalized multi-walled carbon nanotube (MWCNT) Buckypaper/Polyvinyl alcohol (BP/PVA) membrane and employed for degradation of methylene blue dye. The parameters of the isotherm and kinetic models are estimating using ant colony optimization (ACO), which do not meddle the non-linearity form of the respective models. The proposed inverse modelling through ACO optimization was implemented, and the parameters were evaluated to minimize the non-linear error functions. The adsorption of MB dye onto JP-immobilized BP/PVA membrane follows Freundlich isotherm model (R2 = 0.99) and the pseudo 1st order or 2nd kinetic model (R2 = 0.980 & 0.968 respectively). The model predictions from the parameters estimated by ACO resulted values close the experimental values, thus inferring that this approach captured the inherent characteristics of MB adsorption. Moreover, the thermodynamic studies indicated that the adsorption was favourable, spontaneous, and exothermic in nature. The comprehensive structural analyses have confirmed the successful binding of peroxidase onto BP/PVA membrane, as well as the effective MB dye removal using immobilized JP membrane. Compared to BP/PVA membrane, the reusability test revealed that JP-immobilized BP/PVA membrane has better dye removal performances as it can retain 64% of its dye removal efficiency even after eight consecutive cycles. Therefore, the experimental results along with modelling results demonstrated that JP-immobilized BP/PVA membrane is expected to bring notable impacts for the development of effective green and sustainable wastewater treatment technologies.
Modeling and optimization by particle swarm embedded neural network for adsorption of methylene blue by jicama peroxidase immobilized on buckypaper/polyvinyl alcohol membrane

Jun LY, Karri RR, Yon LS, Mubarak NM, Bing CH, Mohammad K, et al.

Environ Res, 2020 04;183:109158.
PMID: 32044575 DOI: 10.1016/j.envres.2020.109158

Jicama peroxidase (JP) immobilized functionalized Buckypaper/Polyvinyl alcohol (BP/PVA) membrane was synthesized and evaluated as a promising nanobiocomposite membrane for methylene blue (MB) dye removal from aqueous solution. The effects of independent process variables, including pH, agitation speed, initial concentration of hydrogen peroxide (H2O2), and contact time on dye removal efficiency were investigated systematically. Both Response Surface Methodology (RSM) and Artificial Neural Network coupled with Particle Swarm Optimization (ANN-PSO) approaches were used for predicting the optimum process parameters to achieve maximum MB dye removal efficiency. The best optimal topology for PSO embedded ANN architecture was found to be 4-6-1. This optimized network provided higher R2 values for randomized training, testing and validation data sets, which are 0.944, 0.931 and 0.946 respectively, thus confirming the efficacy of the ANN-PSO model. Compared to RSM, results confirmed that the hybrid ANN-PSO shows superior modeling capability for prediction of MB dye removal. The maximum MB dye removal efficiency of 99.5% was achieved at pH-5.77, 179 rpm, ratio of H2O2/MB dye of 73.2:1, within 229 min. Thus, this work demonstrated that JP-immobilized BP/PVA membrane is a promising and feasible alternative for treating industrial effluent.