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Summary of Group Research

Our group has research projects in biomolecular related areas. These are summarized below.

1 Biomimetic Materials and Devices
(I) Tissue Engineering
What are the limitations to current tissue engineering systems? Recent strategies of engineering an organ in vitro starts with a biodegradable polymer scaffold designed to simulate the extracellular environment of the body. Autologous or allogenous cells would then be seeded onto the scaffold to proliferate and differentiate. Finally, the cell-polymer construct would be incubated within a bioreactor until a form suitable for implantation is achieved. However, there are many problems encountered to date with this approach, such as: (a) sizes of scaffold being small due to difficulties in seeding cells, in vascularization and in internal necrosis; (b) dedifferentiation of cells after seeding onto the scaffold due to loss of cell-cell contact, extracellular matrix and dissolved growth factors; (c) the inability of random culture of mixed cell population to organize and form a complex tissue; and (d) the poor integration of the engineered tissue into the host organ.

It is my unique strategy, and the core hypothesis of our group since I started at NUS in 2001, that the use of microspheres as scaffolds can overcome all of the limitations above by mimicking cell development at the embryonic stage. As the embryo forms a microspherical blastocyst, cells provide 3-dimensional contact and biochemical cues to differentiate and proliferate. We have found, as have been published in our recent works, that polymeric microspheres are indeed very versatile and suitable for complex soft tissues such as the liver. In particular, (i) they can be easily fabricated from different polymers to tailor the degradation rates for the required growth rates of the cells, (ii) their surfaces can be easily modified using a variety of methods to attach peptides, proteins and carbohydrates to control cell adhesion, migration and proliferation, (iii) they can encapsulate various growth factors for controlled release at different time points to regulate cell differentiation and proliferation, and (iv) different types of cells can be cultured on different batches of microspheres for subsequent controlled co-cultures for organization into the required tissue structures. By providing cells with all the environmental cues as found in vivo, we are taking a biomimetic approach where we hypothesize that the cells can re-organize themselves from isolated cells into tissues as they would during the developmental stage.

An MEng student, Chaw Su Thwin, was the original contributor in starting this project in 2001, while a PhD student, Zhu Xinhao, has subsequently laid the foundation of successfully developing a microsphere-tissue construct comprising liver cells and fibroblasts. This was followed by 2 PhD students, Chen Wenhui and Anjaneyulu Kodali. Wenhui grew neuronal cells that showed axonal extension across microspheres, mimicking the nerve cell contacts made in the fetal stage of development; and in the meantime, Anjaneyulu grew adipose derived stem cells on the microspheres to differentiate into bone, fat and liver cells, truly mimicking the embryo development. This combination of adult stem cells with growth factors on microspheres is the culmination of our hypothesis, with a publication in Macromolecular Biosciences in 2014 described in section 3.2.1 below. Subsequently, another PhD student, Liang Youyun took this work and combined it with collagen gels to mimic cancer cells, developing an understanding of the stiffness of matrices that control cancer proliferation. This early thrust of my research was funded by my start-up NUS AcRF Tier 1 grant in 2001 that resulted in 5 publications, 7 conference presentations and one invited lecture. There were two follow-up grants from NUS AcRF Tier 1 in 2007 and 2011 that enabled me to continue the work, resulting in 8 publications, 11 conference presentations, 1 keynote lecture (APCChE 2014) and 1 plenary lecture (SMS 2011)

(II) Collagen-mimetic peptides
Following from our work above, I have been keenly interested in designing a biodegradable, biocompatible and biomimetic material to be used either to form the microspheres or as gels to hold the microspheres together. Arising from this is a collaborative project with Prof Michael Raghunath and Prof Li Jun (both from the Division of Bioengineering, NUS) in which a joint group project within the Faculty of Engineering was started through the joint funding of 3 NUS AcRF Tier 1 grants in 2005. This project was first lead by a PhD student, Khew Shih Tak, in which a biomimetic collagen was designed firstly using peptides and subsequently by coupling with dendrimers. The culmination of these works resulted in the design of a material that can behave like collagen but without the drawbacks of natural collagen. The results of the work have been published in 6 papers, and also presented in 7 conference papers.

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Figure 3. Adipose derived stem cells growing on gelatin microspheres (I), and highly organized bundles of CMPA fibrils (II).

The initial work lead to an interesting idea of better mimicking collagen using peptide amphiphiles. By combining a bioactive collagen-mimicking portion of the peptide amphiphile with self-assembling portions, we have developed a material which very closely mimics collagen, both biologically and physically. Cell adhesion and detachment studies have shown a 99% mimicry of the collagen mimetic peptide amphiphiles (CMPA) to natural collagen, while self-assembly process shows the physical mimicry of collagen fibrils and bundles. This work was initially done by a PhD student, Luo Jingnan, with a seminal paper in ACS Nano in 2011, described in more detail in section 3.2.1. I was also invited for a keynote lecture in the 9th World Congress of Chemical Engineering in 2013 for this work. Following from these, another 2 students, Chen Yiren and Sushmita Sundar, also applied the use of CMPAs to further mimic elastin and fibronectin, developing the biomimetic peptides into a solution that gels on demand within 5-10 seconds. This was funded through a project with Michael Raghunath in 2009, and another with Jiang Jianwen in 2012. Overall, we had 2 publications and 8 conference presentations from this work.

(III) Biomimetic Devices
In recent years, my interest in tissue engineering has shifted from growing replacement organs to forming small tissue pieces as we learnt about the complexities of tissue organization and growth into complete organs. As we have shown to be successful in constructing tissue-like cells on microspheres, I have started a group at NUS in 2015 to develop organs-on-a-chip with colleagues from NUS (Saif Khan, Chen Chia-Hung, David Leong, Lim Thiam Chye), NTU (Sierin Lim, Kang Yuejun), and UIUC (Kong Hyun-Joon), with other potential collaborators like Samir Mitragotri (Harvard) and Alireza Khademhosseini (MIT). Our strategy is to build an interlinked-system of different tissues, mimicking the connectivity of different organs with 2-dimensional tissue pieces. As this is a relatively new initiative, we have been applying for grants to support this research since 2016.

Figure 4. Preliminary design of interlinked organs-on-a-chip to mimic body function.

2 Biomimetic Membranes
In the area of biomimetic membranes, the original research started with molecular imprinting to mimic antibodies. Subsequently with our success in imprinting proteins onto particles, we developed molecularly imprinted membranes for continuous protein separation. This eventually lead to the idea of mimicking cellular membranes using proteins, which is one of our best works in the field of biomimetic membranes.

(I) Molecular Imprinting
The recognition of any molecule is one of Mother Nature’s secret behind the monitoring and co-ordination of various reactions and activities, like respiration, reproduction, and antibody recognition. Molecular imprinting is currently thought of as a feasible technique in imparting molecular recognition properties to synthetic materials. This is a technique which involves the formation of binding sites in a synthetic polymer matrix that are of complementary functional and structural character to its ‘substrate’ molecule. Conventionally, molecular imprinting is carried out through the preparation of a bulky imprinted polymer using the non-covalent approach. However, there are limitations to this method. Firstly, bulk imprinting produces imprinted polymer of sharp and irregular shape which limits its application. Secondly, it is only limited to small template molecules as it will not be easy for the macromolecules to diffuse through to reach the binding sites during rebinding. Thirdly, due to the fragile and sensitive nature of proteins, it is not easy to achieve a good imprinting effect for protein molecules since the reaction environment can easily denature and unfold the template molecules. Lastly, although bulk imprinting is easily carried out in the laboratory, this process is not suitable to be employed at the industrial scale due to its poor thermal dispersion.

I have started research in 2004 in molecular imprinting of proteins for three reasons. The main reason is due to my interest in using growth factors for tissue engineering as above, and a major problem is in obtaining sufficient quantities at low cost for our use. The high cost of proteins including growth factors is mostly due to the purification steps, and having knowledge of molecular imprinting, I believed that this method could be used to lower the production cost. Secondly, with our skills developed from fabricating microspheres, I hypothesized that using polymeric nanoparticles can significantly enhance the success of large molecule imprinting, eliminating the limitations of conventional imprinting as described above. Finally, Prof Bai Renbi (then the Division of Environmental Science and Engineering, NUS) had interest in protein adsorption and had invited me to collaborate in a project on protein separation supported by an NUS AcRF grant. Lead by my PhD student, Tan Chau Jin, we have fabricated polymeric nanoparticles that have shown an unprecedented ability to selectively adsorb the template protein from a mixture of proteins in aqueous solution. The results for this work have resulted in 5 publications, 3 conference presentations, 2 invention disclosures leading to one patent granted in 2009. Our success in molecularly imprinted nanoparticles is the first in the world for highly selective protein separation in a complex aqueous mixture, and the leading paper, discussed in section 3.2.1, published in Analytical Chemistry in 2008 is among my best work to date.

Arising from this, we subsequently were awarded a Bill & Melinda Gates Foundation Exploratory Grant in their first call in 2009 to develop viral imprinted nanoparticles for prevention in virus infection. Together with a PhD student, Niranjani Sankarakumar, we showed that our particles can hinder the infection of E. coli bacteria by M13 bacteriophages, mimicking the capture of viruses by antibodies or white blood cells in nature. This is the first time ever that a polymeric particle was shown to catch viruses, even in a bacteria model, to stop their infection of cells. The resulting 2 publications and 6 conference presentations. In 2014, I collaborated with Prof Christina Chai (Dept of Pharmacy, NUS) to develop these molecularly imprinted nanoparticles for the detection of algal metabolites and toxins, through a grant from NRF EWI. We have filed an invention disclosure in 2017 and were awarded a provisional patent for the MIPs, receiving interest from one of the largest analytical companies, Shimadzu Inc., to commercialize the technology.

Figure 5. Molecularly imprinted nanoparticles for protein separation (I) and aquaporin biomimetic membranes (II).

(II) Aquaporin Biomimetic Membranes
Our skills in molecularly imprinting proteins onto polymeric nanoparticles have lead to ideas on molecularly imprinting proteins onto membranes for continuous separation. In an unpublished work in 2009 by a PhD student, Wang Honglei, she demonstrated that these protein imprinted membranes can specifically separate out a two-protein mixture. As Honglei was co-supervised by my colleague in ChBE, Prof Neal Chung, one of the top 3 membrane experts in the world, he suggested a collaboration with him and another colleague from the Department of Biochemistry, Prof Jeyaseelan Kandiah, on using aquaporins imprinted onto membranes for water purification.

Aquaporins are transmembrane water channels that exists in all living cells, from single cell bacteria to eukaryotic cells like mammals and humans. First discovered by Peter Agre in 1994, for which he won the Nobel Prize, aquaporins have been found to allow only water molecules to pass through and at a very high rate. Therefore, many have theorized that aquaporins can be used as water channels in synthetic water purification membranes for seawater desalination, potable water purification and even wastewater treatment. However, no research lab or commercial company has successfully developed such a membrane that mimics cells. I formulated a strategy to use polymeric and lipid vesicles with aquaporins incorporated into the vesicle membranes in a way to mimic the way mangrove tree filters seawater with its root cells. Instead of using living cells, the vesicles behave like cells, allowing only water to permeate, while an impermeable barrier prevents salts from entering (Figure 5(II)). We were the first group in the world to thus successfully fabricate such a biomimetic membrane with aquaporins, having the highest flux of pure water that is 10 times more than commercial reverse osmosis membranes. After applying for a patent in 2012, we published this method and membrane in the Journal of Materials Chemistry A in 2013. From an initial grant in 2009 by the NRF Environment and Water Industry Programme Office (EWI) for $3.7 million, this work lead to a subsequent larger grant of $8.8 million in 2012 to improve the membrane, scale-up the production of both the membranes and aquaporins, and test the stability of the membranes in various environmental conditions. In this large collaborative project, my own group published 11 out of the 15 papers with 2 patents awarded. We are now discussing the licensing of the patent with a company in China, AQ Biomimic Ltd while also preparing our own spin-off company to produce aquaporins at large scales. I was also invited to give a plenary lecture at the International Conference on Soft Materials (ICSM 2014) in Jaipur, India in 2014 for our work in biomimetic membranes.

3 Biomimetic Systems
My third major research area in biomimetics started from a relatively unusual idea of growing bacterial cells on our polymeric microspheres, earlier described in section 3.1.3. In 2011 colleague in ChBE, Prof Loh Kai-Chee, decided to collaborate on biological transformation of chemicals for applications in the pharmaceutical industry as a more environmentally friendlier way of producing active pharmaceutical ingredients. At that time, GSK Singapore partnered with the Singapore Economic Development Board (EDB) to launch a grant call for Green and Sustainable Manufacturing, so we submitted a proposal using microspheres and membranes to encapsulate or carry bacteria cells for a class of small molecular hydroxylations. With the award of this grant, my Research Fellow, Wang Liang, grew Sphingomonas bacteria onto PHBV microspheres to effectively hydroxylate pyrrolidine. Another Research Fellow, Cheng Xi-Yu, managed to encapsulate Pseudonomonas bacteria in hollow fiber membranes to hydroxylate indene. These two works, published in 2014 in the Journal of Biotechnology and the Biochemical Engineering Journal respectively, mimics the way nature protects bacteria cells with biofilms to allow cells to survive in environments that are usually toxic, with high concentrations of organics, and effectively use these chemicals as substrates for growth.

This initial project lead my interest into the area of microbial transformation. In 2012, I participated in the preparation of a large collaboration between NUS and Shanghai Jiaotong University (SJTU), looking into coupled challenges within megacities of Singapore and Shanghai. One major challenge was in waste management, and I proposed that we could reduce the footprint for treatment of food wastes, and wet organic wastes like yard wastes or sewage wastes, using microbial transformation, in particular anaerobic digestion (AD). From initially being the PI of one project within this major programme, I was asked to help collate all of the projects, and eventually took over the role to lead in writing the full proposal the entire programme, which was awarded in 2012 for $24 million.

(I) Biomimetic AD Systems
This programme, called the Energy and Environmental Sustainability Solutions for Megacities (E2S2-CREATE), is funded under the NRF Campus for Research Excellence and Technological Enterprise for 5 years until 2017. With 30 professors each from NUS and SJTU, we undertook research in waste management and emerging contaminants for a more sustainable city. I lead the team in waste management, with 10 professors, 8 research fellows and 12 PhD students, studying all aspects starting from waste disposal behavior to collection methods, from biological to chemical technologies to convert wastes into energy, and from power generation and heat reutilization. My own particular focus within the programme was in the anaerobic digestion of wet organic waste fraction of the municipal solid waste mixture. Our initial research found that current methods of waste treatment, which handles this complex mixture as a whole, is very inefficient, for example in producing energy from incineration but also leaves a high volume of residue as bottom ash. Therefore, we proposed to separate the dry and wet fraction of organic wastes for thermochemical and biological treatment respectively, and recycling the inorganic components.

With a multitude of biological treatment methods, including composting and aerobic digestion, I proposed the use of high solid AD to be more suitable for cities, especially for decentralized treatment at source for food wastes. High solid AD systems are expected to be more compact and socially acceptable for placement in cities, and would be able to convert food waste into methane for power generation. With this goal, our group has designed a novel 3-stage anaerobic digester that mimics the stomach of cows, where the first stage acts to hydrolyze wastes, the second stage converts the hydrolyzed compounds into acids and acetate, and the third stage produces methane with anaerobes (Figure 6(I)). We found that this system has a higher biogas production and a shorter digestion time while maintaining consistent operation over a period of 1 year, with a paper published in Scientific Reports in 2017. At the same time, we studied waste pretreatment, co-digestion of food wastes with manure or horticultural wastes, the effects of plastics in the waste streams, energy performances of AD, combination of AD with gasification for combined waste treatment, and even the microbial community composition and manipulation. To date, we have 6 papers on AD by our group with another 3 in press, and 3 more submitted. Together with Prof Wang Chi-Hwa, our combined studies in AD and gasification resulted in 7 papers and 2 submitted. We have also collaborative projects with the National Environment Agency to model food waste treatment in hawker centers throughout Singapore, and with 3 companies on industrial testing of digesters and gasifiers. For this work, I was awarded the Dr G.P. Kane Chemcon Distinguished Speaker Award by the Indian Institute of Chemical Engineers in 2014, giving a plenary lecture at their ChemCon 2014 in Chandigarh, India.

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Figure 6. Our patented 3-stage anaerobic digester for food waste digestion (I). Algal photobioreactors reducing nitrogen and phosphorus in AD discharge (II).

(II) Biomimetic Photobioreactors Systems
During the study of anaerobic digestion of organic materials, we discovered that the residual sludge from the digester contains a high amount of nitrogen and phosphorus. The AD process also generates a significant amount of carbon dioxide in the biogas together with methane, with CO2 amounts ranging from 25-40% of the total biogas. In an effort to reduce both the CO2 and aqueous emissions, we studied the use of algae in a novel photobioreactor system. In this design, hollow fibers are used to interconnect the output sludge with the photobioreactor, supplying algae with CO2 while oxygen is supplied to the sludge. This mimics the symbiotic growth of bacteria and algae in nature while being separated in individual reactors. We found a large increase with algae biomass yield and CO2 reduction as compared to conventional photobioreactors, and almost complete removal of nitrogen and phosphorus. One key paper in this symbiotic study was published in the Journal of Applied Phycology in 2014, with another 3 related algal studies also published.

4 Collaborative Research
Polymeric drug and gene delivery
The last thrust of my research is mainly an outcome of the need to incorporate controlled growth factor delivery into our biomimetic materials for tissue engineering and organ-on-a-chip devices. In this regard, I have been collaborating with Prof Wang Chi-Hwa (ChBE, NUS) and Dr Yang Yiyan (Institute of Bioengineering and Nanotechnology (IBN), A*STAR, Singapore) to learn about drug and gene delivery since 2001. Prof Wang is an expert in drug delivery for liver and brain cancer, with many publications on modeling drug release from micro- and nanoparticles. We co-supervised my PhD student, Zhu Xinhao, on integrating growth factor release from the polymeric microspheres. In addition to the tissue engineering applications as described above, we had also co-supervised a post-doctoral fellow, Dr Hu Yong, resulting in 2 publications on controlled drug release and their effects on hepatoma cells. We subsequently co-supervised another PhD student, Yan Weicheng, in 2013 who fabricated core-shell microparticles using electrohydrodynamic atomization for stroke treatment, who has been very productive with 6 publications to date.

The second collaboration, with Dr Yang, also started when I joined NUS in 2001. I collaborated with Dr Yang to co-supervise my very first PhD student, Liu Shaoqiong, on synthesizing stimuli-sensitive nanoparticles for cancer treatment. In return, I had then co-supervised Dr Yang’s MEng student, Chooi Kar Wai. As my expertise was in polymer synthesis, I acted as the main adviser for both students on the synthesis of PNIPAAm and its copolymers, while Dr Yang provided the funding, equipment and other resources. Through this collaboration, we have developed knowledge on stimuli-controlled drug and gene delivery, with the ability to characterize nanoparticles, their drug encapsulation strategies, and their effects on cells. The results were published in 5 papers (in which I agreed to be only the co-author for funding purposes) and presented in 3 conferences. We continued our strong collaboration since then, co-supervising 3 PhD students, Nikken Wiradharma from 2004 to 2009, Ke Xiyu and Willy Chin from 2011 until 2017. Our work expanded into peptide-mimetic drug delivery for anticancer treatment with Nikken (with 4 papers), polymeric micelles co-delivery of multiple drugs by Xiyu (2 papers), and antimicrobial polycarbonates by Willy (2 papers).