USE OF UPCONVERSION FLUORESCENCE NANOPARTICLES IN BIOMEDICAL APPLICATIONS DEV KUMAR CHATTERJEE (M.B.B.S., M.M.S.T) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DIVISION OF BIOENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2007 _______________________________________________________________________________ PREFACE This thesis is hereby submitted for the degree of Doctor of Philosophy in the Division of Bioengineering at the Faculty of Engineering, National University of Singapore. This thesis, either in part or whole, has never been submitted for any other degree or equivalent to another university or institution. This thesis contains all original work, unless specifically mentioned and referenced to other works. Parts of this thesis has been published or presented in: Peer reviewed journal publications: Chatterjee, D.K., Fong L.S., Zhang Y., Nanoparticles in photodynamic therapy: an emerging paradigm. Advanced Drug Delivery Reviews (Invited article, under review) Chatterjee, D.K., Zhang, Y. Upconverting Nanoparticles as Nano-Transducers for Photodynamic Therapy in Cancer Cells, Nanomedicine Vol. 3, No. (2008) 73-82. Chatterjee, D.K., Zhang, Y. Upconversion fluorescence imaging of cells and small animals using lanthanide doped nanocrystals, Biomaterials Volume 29, Issue 7, (2008) 937-943 Wang, F., Chatterjee, D.K., Li, Z.Q., Zhang, Y., Fan, X.P., Wang, M.Q. Synthesis of polyethylenimine/NaYF4 nanoparticles with upconversion fluorescence, Nanotechnology, vol. 17, No 23 (14 December 2006) 5786-5791 ii _______________________________________________________________________________ Review book chapters Chatterjee D.K. and Zhang, Y. (2007). Lanthanide doped Upconverting Nanoparticles for biomedical applications. Doped Nanomaterial and Nanodevices. Wei Chen. American Scientific Publishers (in press). Chatterjee, D.K. and Y. Zhang (2007). Nanoparticles in Immunotherapy Against Cancer. Cancer Nanotechnology – Nanomaterials for Cancer Diagnosis and Therapy. H. S. Nalwa and T. Webster. Valencia, American Scientific Publishers. 317 - 332 Zhang, Y. and Chatterjee D.K. (2006). Liposomes, dendrimers and other polymeric nanoparticles for targeted delivery of anticancer agents - A comparative study. Nanomaterials for Cancer Therapy. C. S. S. R. Kumar. Weinheim, Wiley-VCH Verlag GmbH & Co, KGaA. 6: 338 - 370. Conference abstracts: Chatterjee DK and Zhang Y, Upconverting Nanoparticles as Nano-Transducers for Photodynamic Therapy in Cancer Cells. (NSTI Nanotechnology Conference and Trade Show, June 1-5, 2008, in Boston, Massachusetts, U.S.A) Chatterjee DK and Zhang Y, Upconverting Nanoparticles for in vitro and in vivo imaging. 2008 (NSTI Nanotechnology Conference and Trade Show, June 1-5, 2008, in Boston, Massachusetts, U.S.A) iii _______________________________________________________________________________ Chatterjee D.K. and Zhang Y., Up-converting Nanoparticles: Novel Soluble Probes for Imaging of Live Cancer Cells and Tissues. 2007 Spring Proceedings; Volume 1019E 1019-FF08-11 (2007). Moscone West: San Francisco Marriott, San Francisco, CA, USA (2007 MRS Spring Meeting, April 9—13, 2007) Zhang Y and Chatterjee DK, Multi-functional nanoparticles for cancer therapy. Abstract Book of International Symposium on Nanotechnology in Environmental Protection and Pollution (2006): 31. Hong Kong: The Hong Kong University of Science & Technology .(International Symposium on Nanotechnology in Environmental Protection and Pollution, 18 -21 Jun 2006, The Hong Kong University of Science & Technology, Hong Kong, China) Chatterjee, D.K., Zhang, Y. Multi-functional nanoparticles for cancer therapy, Science and Technology of Advanced Materials, vol (2007) 131-133 Chatterjee DK and Zhang Y, Evaluation of the biocompatibility of the bi-functional nanoparticles. Proceedings of The 12th International Conference on Biomedical Engineering (2005). Singapore: IFMBE. (The 12th International Conference on Biomedical Engineering (ICBME 2005), - 10 Dec 2005, Singapore) Chatterjee DK and Zhang Y, Synthesis and Characterization of Bi-functional Nanoparticles for Cancer Immunotherapy. Proceedings of The 12th International Conference on Biomedical Engineering (2005). Singapore: IFMBE. (The 12th International Conference on Biomedical Engineering (ICBME 2005), - 10 Dec 2005, Singapore) iv _______________________________________________________________________________ ACKNOWLEDGEMENTS I would like to acknowledge the contributions of my guide A/Prof Zhang Yong for his constant encouragement, guidance and advice without which none of this would have been possible. I have also been supported during this long effort by my colleagues who have taught me procedures or helped with the synthesis of the nanoparticles. The help from Initha Appavoo, Dr Li Zhengquan (nanoparticles) and Dr Rufaihah (animal experiments) deserve a special mention. A special note of thanks to those undergraduates – primarily Lim Sock Yong, Xiuli and Eliza – who have put in long hard hours and challenged me with their constant queries. All have contributed to make this journey not only a learning one but also an enjoyable one. I would also like to acknowledge the research grant from the National University of Singapore for the essential financial support. Finally, my thanks to my family - and especially my wife Deyali - whose constant love and support helped me through the toughest times. Dev Kumar Chatterjee April, 2008 v _______________________________________________________________________________ TABLE OF CONTENTS PREFACE . II ACKNOWLEDGEMENTS V TABLE OF CONTENTS . VI SUMMARY IX LIST OF TABLES . XII LIST OF FIGURES XIII ABBREVIATIONS . XVI CHAPTER LITERATURE REVIEW & RESEARCH PROGRAM . 1.1 Definition and scope . 1.2 Nanoparticles for disease diagnostics 1.2.1 Molecular targeting using nanoparticles . 1.2.2 Fluorescent nanoparticles as imaging probes 1.3 Nanoparticles in therapeutic applications . 16 1.3.1 General principles 16 1.3.2 Nanoparticles for photodynamic therapy of cancer . 19 1.4 Upconversion nanoparticles . 26 1.4.1 Principle of upconversion 26 1.4.2 Upconversion nanoparticles: definition and materials . 29 1.4.3 Surface modifications of upconverting nanoparticles . 32 1.5 Thesis overview 36 CHAPTER SYNTHESIS & CHARACTERIZATION OF UPCONVERSION NANOPARTICLES . 40 2.1 Introduction 41 2.2 Materials and Methods 44 2.2.1 Reagents 44 2.2.2 Synthesis of PEI/NaYF4 nanoparticles . 45 2.2.3 Physical characterization of the nanoparticles 45 2.2.4 Optical characterization . 48 vi _______________________________________________________________________________ 2.2.5 Cell biocompatibility test . 52 2.3 Results and Discussion . 55 2.3.1 Physical characterization of the nanoparticles 55 2.3.2 Optical characterization of the nanoparticles 61 2.3.3 Cell viability test 73 2.4 Conclusion 78 CHAPTER IMAGING OF CANCER CELLS USING UPCONVERSION NANOPARTICLES . 79 3.1 Introduction 80 3.2 Materials and Methods 81 3.2.1 Materials 81 3.2.2 Attachment of targeting ligand on upconversion nanoparticles . 81 3.2.3 Size measurement with TEM . 82 3.2.4 Surface charge measurement 82 3.2.5 Detection of aggregates in solution 83 3.2.6 Detection of folic acid on the nanoparticles 83 3.2.7 Incubation of nanoparticles with cancer cells . 84 3.2.8 Confocal imaging . 84 3.2.9 Efficiency and specificity of targeting of nanoparticles to cancer cells 87 3.3 Results . 88 3.3.1 TEM of FA-PEI/NaYF4 . 88 3.3.2 Confirmation of folic acid binding on nanoparticles by FTIR . 89 3.3.3 Alteration of zeta-potential due to folic acid attachment . 90 3.3.4 Detection of aggregates by size distribution . 90 3.3.5 Imaging of cancer cells 91 3.3.6 Effect of incubation period on uptake of nanoparticles . 94 3.4 Conclusion 99 CHAPTER UPCONVERSION NANOPARTICLES FOR IN VIVO IMAGING 100 4.1 Introduction 101 4.2 Materials and Methods 104 4.2.1 Materials 104 4.2.2 Imaging of upconversion nanoparticles within rat skin . 105 4.2.3 Comparison of upconversion nanoparticles with QDs for in vivo imaging 105 4.2.4 Imaging of upconversion nanoparticles in other rat tissues . 106 4.2.5 In vivo microscopy using upconversion nanoparticles 106 4.3 Results and Discussion . 109 4.3.1 Imaging of subcutaneously injected upconversion nanoparticles vii _______________________________________________________________________________ . 109 4.3.2 Comparative imaging of subcutaneous injection of nanoparticles 111 4.3.3 Imaging of injected nanoparticles in other tissues . 112 4.3.4 In vivo cell imaging . 113 4.4. Conclusion . 116 CHAPTER UPCONVERSION NANOPARTICLES IN PHOTODYNAMIC THERAPY OF CANCER .117 5.1 Introduction 118 5.2 Materials and Methods 120 5.2.1 Materials 120 5.2.2 Preparation of ZnPC standard curve by spectrophotometry 121 5.2.3 Attaching ZnPC to FA-PEI/NaYF4:Yb,Er nanoparticles 121 5.2.4 Detection of ZnPC on the surface of the nanoparticles . 123 5.2.5 Determination of singlet oxygen production . 123 5.2.6 Targeted binding to human cancer cells 124 5.2.7 Photoexposure of cells . 124 5.2.8 MTT assay to check effectiveness of PDT 124 5.3 Results and Discussion . 126 5.3.1 Standard curve for ZnPC 126 5.3.2. Encapsulation efficiency . 126 5.3.3 FTIR for presence of ZnPC 127 5.3.4 Spectroscopy to determine emission-excitation overlap 128 5.3.5 Singlet oxygen production by ADPA molecular probe 129 5.3.6 Targeted uptake of ZnPC-UCN by cancer cells 131 5.3.7 Effectiveness of PDT using ZnPC-UCN . 132 5.3.8 Effect of nanoparticle concentration . 134 5.4 Conclusion 135 CHAPTER CONCLUSION AND FUTURE WORK 137 REFERENCES 142 viii _______________________________________________________________________________ SUMMARY Nanoparticles are spherical aggregates less than 100nm in diameter containing a few hundreds to thousands of atoms. Fluorescent nanoparticles excited by near infrared (NIR) are advantageous because NIR gives rise to minimal autofluorescence which results in very high signal-to-background ratios; cells and tissue destruction is low because NIR is harmless to biomolecules in low doses; and nanoparticles can be imaged from inside tissues because of deep penetration of NIR radiation. This thesis explores the characterization and biomedical applications of a new variety of NIR excited fluorescent nanoparticles, PEI/NaYF44:Er,Yb, developed at the Cellular and Molecular Bioengineering Laboratory, with a focus on cancer. PEI/NaYF4 upconversion nanoparticles co-doped with Er and Yb were demonstrated to be 60 nm spherical particles of uniform shape and size, positive surface charge and stably soluble in de-ionized water. When excited with 980nm laser these emitted light with sharp peaks in the red and green region of the visible spectrum. This emission was strongly photostable and immune to storage over weeks, although incubation in serum at physiological temperatures slowly degraded the signal, probably by protein deposition. The particles were biocompatible with two different human cell lines to moderately high concentrations and for reasonable periods of incubation. The upconverting nanoparticles (UCN) were conjugated to a cell-specific ligand and used for targeted imaging of live human cancer cells in vitro. These showed strong signal-to-background ratios and high sensitivity of detection. Non-targeted tagging of cells using PEI polymer as a positively charged coupler was also demonstrated. All imaging experiments showed signal stability and absence of cell damage as a result of ix _______________________________________________________________________________ prolonged laser exposure. The ability to image these nanoparticles inside animals was demonstrated by injecting into various tissues of live, anaesthetized rats and exciting the injection site with NIR laser. Fluorescence from injected nanoparticles was recorded at injection depths of a few millimeters to nearly cm, the depth depending on the type of tissue injected, the dose of nanoparticles and the effective control of ambient light which contributes to background. Human cancer cells, non-specifically tagged with upconversion nanoparticles, were injected subcutaneously in live anaesthetized mice and the cells imaged by real-time in vivo confocal microscopy. Photodynamic therapy (PDT) is a therapeutic option for cancer that relies on the interaction of light and photosensitizer drugs to kill targeted cells. Acceptance of PDT has been limited by, among other factors, fear of high cost of setup and inability to easily reach deeper seated tumors. We demonstrated a nanoparticle-based approach to address these problems. UCN were functionalized with zinc phthalocyanine (ZnPC) photosensitizer for simultaneous imaging and photodynamic therapy. The nanoparticles act as ‘nano-transducers’ to convert deeply tissue penetrating NIR excitation to emission frequencies suitable to activate the photosensitizer to release reactive oxygen species to kill cancer cells. The effectiveness of the modified nanoparticles for this purpose was demonstrated in vitro with concurrent imaging. Both the imaging and photodynamic therapy of cancer cells using PEI/NaYF44:Er,Yb nanoparticles were described, to the best of my knowledge, for the first time, although several preliminary results using large phosphor ‘nanoparticles’ in excess of 100nm in diameter can be found in the literature. These results lead the slow emergence of the phosphor nanoparticles as a valuable fluorescent label for biomedical applications, set x _______________________________________________________________________________ had been demonstrated that coating with folic acid promoted selectivity of binding to cancer cells over normal cells (e.g. fibroblasts). Hence, it can be reasonably assumed that the FA-ZnPC-UCN demonstrated here will also show selectivity towards cancer cells. In conclusion, we have described a nanodevice for cancer PDT where upconverting nanoparticles convert deeply penetrating NIR light to visible emissions, which are in turn used by the attached photosensitizer to convert molecular oxygen to toxic singlet species. It can be appreciated that this is essentially a platform technology with the scope to substitute ZnPC with other photosensitizers which absorb in the 540nm or 650nm range. For example, tin etiopurpurin (SnET2) requires 660nm while tetra(mhydroxyphenyl)chlorine (mTHPC, Foscan) is excited at 652nm. Further studies into effectiveness of this therapy in animals are being explored. 136 Chapter CONCLUSION AND FUTURE WORK _______________________________________________________________________________ Fluorescent labels that are excited by the near infrared (NIR) radiation give rise to minimal autofluorescence because of lack of efficient endogenous absorbers in the NIR spectral range (Bremer et al., 2001; Konig, 2000; Larson et al., 2003) and the absence of upconversion among most biomolecules. This results in very high signal to background ratios. Photo-damage to cells and tissues is also greatly reduced because NIR is generally harmless to biomolecules in low doses. Penetration depths are increased several orders of magnitude (from a few mm for UV to up to as deep as 15 cm in fibrofatty tissues like breast tissues for NIR). (Auzel, 2004; Soukka et al., 2005). The rare earth elements used in synthesis of upconversion nanoparticles have a lower toxicity than semiconductor elements of quantum dots. These chemically stable particles not bleach and allow permanent excitation with simultaneous signal integration (Corstjens et al., 2005). Since upconversion occurs within the host crystal, optical properties are unaffected by the environment (e.g., chemicals, pH and temperature). Consequently, use of these particles is unaffected by the nature of the analyte in assays (such as whole blood, plasma, urine, sputum, or tissue homogenates) or by the microenvironment in cellular and subcellular imaging. UCNs typically have narrower absorption and line emission spectra, as compared to the downconverting fluorophores. The emission of upconverting particles consist of sharp lines characteristic of atomic transitions in a well-ordered matrix. To exploit these advantages, upconverting nanoparticles, or UCN, need to be developed and explored. For cancer cell targeting, folic acid was covalently conjugated to the PEI/NaYF4 nanoparticles. The method of cross-linking FA to PEI involves a simple condensation reaction and the substitution of folic acid by other biomolecules such as antibodies or peptides can be carried out without significant alteration of the synthesis protocol. In 138 _______________________________________________________________________________ these experiments the human colonic adenocarcinoma cell line HT29 and the ovarian cancer cell line OVCAR3 were used because they possess high amounts of folate receptors on their surface. Live cells were imaged and the images showed the attachment of the upconversion nanoparticles onto the surface of the cells. While the binding of the nanoparticles to the surface of the cells is rapid and extensive fluorescence can be detected within an hour, uptake inside cells through the internalization of the bound receptor-nanoparticle complex is a much slower process and can usually be detected in significant amount only after 48 hours of incubation. However, using NIR to image cells has a disadvantage too. Since the maximum resolution achievable with microscope varies inversely with the wavelength of light used, using NIR imposes a theoretical limit on the quality of the achieved images. This can be seen in the fluorescent images obtained where small bright clusters of the UCN appear to be diffuse masses of nanoparticles. However, in the tradeoff between resolution and sensitivity, NIR presents reasonably high values of both. Uncoated nanoparticles are covered with a surface layer of PEI which is a highly positively charged molecule with a large number of amino groups. It is to be expected that these nanoparticles will be involved in formation of hydrogen bonds with negatively charged molecules on the surface of the cells and also on the free surface of the well plate. These nanoparticles thus show a moderately high non-specific binding to cell surfaces after incubation. Coating with folic acid, however, significantly lowers the amount of non-specific binding and spurious background luminescence can be avoided with thorough washing. 139 _______________________________________________________________________________ However, much can be done to maximize the advantageous use of these nanoparticles in non-invasive live cell imaging. The simplicity of a one-pot synthesis and a universal binding mechanism for any carboxylate group containing ligand lends itself to ease of exploring variations of this theme. Substitutions with antibodies or peptides for more specific targeting can be a step ahead. The reduction of fluorescence degradation in biofluids can be utilized for long term imaging of cellular processes in vitro and in vivo. Addition of other molecules like drugs and biologic response modifiers to confer multi-functional nanodevices is an attractive field of exploration. The animal experiments were carried out using an imaging system equipped with a 980nm laser and simple CCD cameras. Much can be done to improve this setup including mobile imaging platforms to record whole body scans of the animals. Better black-box arrangements can make possible significantly improved signal-to-noise ratios. Laser power can be optimized for maximal luminescence with minimal tissue damage although tissues not have a strong absorption at the wavelength of 980nm. The scope and possible benefits of continuous imaging subcutaneous and muscular tissues in real time is immense and needs to be thoroughly explored. To the best of our knowledge, this represents the first study of imaging of live cells and deep tissues in animal using upconversion fluorescent nanoparticles. Photodynamic therapy of superficial and slightly deeper tumors is an exciting application for these nanoparticles. The next step, the testing of these nanoparticles in small animal model of tumor, has already started in our laboratory. We have started experiments on a bladder and skin tumor model for mice. It is an exciting area of research with potential to significantly affect biomedical diagnostics and therapeutics. 140 _______________________________________________________________________________ In conclusion, UCN present an additional tool to the biologist working with fluorescent nanoparticles. However, in no way these supplant existing fluorescent nanoparticles that have become popular with many biologists and researchers. One of the many reasons for that is the existing fluorescent nanoparticles are very much brighter than the UCN. For nearly all biological experiments, the most important properties of a fluorescent label are brightness and stability. Quantum dots have both qualities. Another advantage of commercially available fluorescent nanoparticles is the much smaller size than UCN. QDs are usually of the order of a few nanometers, usually less than 10nm. This size being similar to that of the average protein molecule, QDs can be used in most biological experiments without hindrance. In contrast, UCN are several times in diameter to QDs and often less bright. While multicolor UCN have been demonstrated, QDs present a larger range of colors mainly because unlike UCN they can tuned by their size and not on the type of impurity. 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Analytical Biochemistry;267(1):30-6. 151 [...]... nanoparticles in tumors is followed by sections which discuss fluorescent nanoparticles used in diagnosis and drug-loaded nanoparticles in therapy 3 _ 1.2.1 Molecular targeting using nanoparticles Definition: Targeting can be loosely defined in this context as any means that increases the specificity of localization of nanoparticles to diseased cells Targeting does not intrinsically... explored biomedical applications of nanoparticles Ferrari (Ferrari, 2005) has dealt with the whole field of cancer nanotechnology, including the in vitro diagnostics as well as in vivo targeting, while Jain has focused on drug delivery in cancer (Jain, 2005) Others have discussed nanotechnology for the biologist (McNeil, 2005) and its uses to the whole field of molecular recognition, mainly for enhanced in. .. either increasing concentration of particles, or by using larger particles or by using higher power of incident radiation The process of upconversion does not necessarily require coherent light, but a focused laser beam is often used in order to obtain high emission efficiency The remarkable photostability of the particles allows the use of laser for relatively long periods without significant bleaching... concentration of the toxic drug while reducing circulating levels of free drug, thus lowering systemic toxicity (Leuschner et al., 2005) Figure 1-1 Different roles of nanoparticles for biomedical applications The following sections deal with each of the three functions separately with specific reference to application in cancer diagnosis and management Description of methods of targeting and concentrating nanoparticles. .. Figure 4-4 Comparing emission from subcutaneous injections of QDs and UCN .111 xiv _ Figure 4-5 Injection into other tissues in Wistar rats: A) muscle and B) heart showed detectable fluorescence 113 Figure 4-6 In vivo live cell imaging using NaYF4 nanoparticles The ‘floating balls’ appearance of the cells in the bottom panel has a distinctive 3-dimensional... Figure 1-2 Tumor targeting with nanoparticles by passive and active targeting 5 Figure 1-3 Principle of photodynamic therapy 20 Figure 1-4 Upconversion involves energy transfer between two excited ion species, resulting in the acceptor ions reaching a higher energy state and subsequently emitting higher energy radiation In contrast, single photon fluorescence has emission of lower energy ... above, the following discussions relate specifically to tumors Passive targeting involves modifications of nanoparticles which increase circulation time without addition of any component/involvement of any method which is specific to the tumor Increased circulation time helps in accumulation of the particles in the tumor by an enhanced permeation and retention, or EPR, effect Long circulating nanoparticles. .. form of special polymer coatings which use steric stabilization The resultant nanoparticles are named ‘stealth’ 5 _ nanoparticles For example, attachment of poly(ethylene glycol) (PEG) ‘hides’ nanoparticles from the MPS enabling longer circulation times Longer time in circulation increases the probability of the nanoparticles being trapped in tumors by EPS In fact,... PEG-coated poly(cyanoacrylate)(pCA) nanoparticles - made by a copolymer inculcating both – has such a long circulating time that they penetrated the brain more than any other modifications, including coating by polysorbate This uptake was increased in pathological situations with presumably higher blood-brain barrier permeability Another example is the incorporation of cisplatin in liposomal formulations (Chawla... with target molecules causes unpredictable changes in the molecular structure of the organic fluorophore resulting in variable reduction in fluorescence conversion efficiency 9 _ Quantum dots (QDs) are nanoparticles of one semiconductor encased in a second semiconductor These can be of considerable use as inorganic fluorophores, because they offer significant advantages . subcutaneous injection of nanoparticles 111 4.3.3 Imaging of injected nanoparticles in other tissues 112 4.3.4 In vivo cell imaging 113 4.4. Conclusion 116 CHAPTER 5 UPCONVERSION NANOPARTICLES IN. Proceedings of The 12th International Conference on Biomedical Engineering (2005). Singapore: IFMBE. (The 12th International Conference on Biomedical Engineering (ICBME 2005), 7 - 10 Dec 2005, Singapore). upconversion nanoparticles with QDs for in vivo imaging 105 4.2.4 Imaging of upconversion nanoparticles in other rat tissues 106 4.2.5 In vivo microscopy using upconversion nanoparticles