Chapter 1 Introduction
1.5 Motivation and Objectives
In most of the research of photothermal cancer therapy, most of them demonstrated metallic nanoparticles or hybrid nanogels containing a metallic core with NIR laser as trigger. Although NIR seems to be easier to penetrate through tissues, the researches using NIR to trigger photothermal effects tends to use a rather higher irradiation intensity. Moreover, NIR applied in the photothermal therapy has been developed rapidly in recent years, but using visual light as energy source is rather rare. As the
reasons mentioned in the former section (section 1.1.2), green laser has been applied in prostatic surgery and has a great outcome. However, it’s hard to find a research paper
using green laser as trigger. Although Wu et. al demonstrated a research using green
laser as light source [26], the carrier was metallic (Au nanoparticles). Even though there’s no research shown the disadvantage of leaving metallic nanoparticles in
physiological surroundings, it was still a concern in long term.
Graphene, carbon-based polymer, has shown special properties and has drown attention in recent years. It shows great photothermal effect and could turn NIR laser -electrons, graphene was also applied in research of photothermal therapy. However, graphene possessed high cytotoxicity, it should be modified to applied in biomedical use.
In this study, we want to use green laser as trigger since it’s visible, easy to operate and high energy density. The objective of this work is to produce a polymeric nanogel as photothermal agent and to induce photothermal effect. We hope this work can shed a light on the application of green laser in photothermal therapy and being effective at a rather low irradiation intensity. We hope this can provide another option in cancer treatments.
Figure 1-1. All cancers (excluding non-melanoma skin cancer) estimated incidence, mortality and prevalence worldwide in 2012, from IARC website.
Figure 1-2. Mechanism of multifunctional MNPs loaded nanofibers [40].
Figure 1-3. TEM image of chitosan-coated Ag nanotriangles [15].
Figure 1-4. Different applications of gold nanoparticles (GNP) in biomedical fields [41].
Figure 1-5. Au nanorod（left）and mesoporous silica coated AuNR (right) [19].
Figure 1-6. Penetration depths of visible and infrared radiation into skin [21].
Figure 1-7. Adsorption coefficient of pure water as measured or compiled by several researchers [27].
Figure 1-8. Nanogels formed with associating polymers and the AFM image [35].
Figure 1-9. Example of pH-responxive hyaluronic nanogels formed with associating polymers .[36]
Figure 1-10. Chemical synthesis of nanogels by copolymerization in colloidal environments. (A)In w/o emulsions. (B) In o/w emulsions [42].
Chapter 2
Materials and Methods
2.1 Chemicals
2.1.1 Preparation and Characteration of Nanogels
A. N-(Dimethylamino) propyl methacrylamide, NDPMA: Cat.#5205-93-6, Sigma-Aldrich, USA
B. Poly (ethylene glycol) diacrylate, PEGDA: Cat.#245801, Sigma-Aldrich, USA
C. Ammonium persulfate, APS: Cat.# 7727-54-0, Sigma, USA
D. Chlorophyllin sodium copper salt, SCC: Cat.# C6003, Sigma-Aldrich E. Span80: Cat.# 71725, Sigma-Aldrich, USA
F. Hexane: Cat.# 110-54-3, Macron
G. Acetone: Cat.#3016-08, RDH Chemical Co.
H. Chlorophyllin sodium copper salt, SCC: Cat.# C6003, Sigma-Aldrich
2.1.2 Cell Culture of Mouse Fibroblast-Like Cell Line L929
A. 10X Trypsin-EDTA (TE): Cat.# T4174, Sigma-Aldrich, USA B. Fetal Bovine Serum (FBS): Cat.# T8153, Sigma-Aldrich, USA C. Alpha minimum essential medium: Cat.# 11900-024, Gibco
D. Gentamicin: Cat.# 15710-064, Gibco
E. Sodium bicarbonate, NaHCO3: Cat.# S7277, Sigma-Aldrich, USA F. Trypan blue: Cat.# T8153, Sigma-Aldrich, USA
G. 2-Mercaptoehanol: Cat.# M3148, Sigma-Aldrich, USA
H. Sodium phosphate dibasic, Na2PO4: Cat.#S0876, Sigma-Aldrich, USA I. Sodium chloride, NaCl: Cat.#4058-01, J. T. Baker, USA
J. Potassium phosphate monobasic, KH2PO4: Cat.# P5655, Sigma-Aldrich, USA
K. Potassium chloride, KCl: Cat.# P9541, Sigma-Aldrich, USA L. Sodium bicarbonate, NaHCO3: Cat.# S7277, Sigma-Aldrich, USA
2.1.3 Cytotoxicity assay (MTS assay)
A. CellTiter 96® AQueous One Solution Reagent: Cat.# G3580 , Promega, USA B. Phenol: Cat.# 108-95-2 Sigma-Aldrich, USA
2.1.4 Cytotoxicity assay (Live/Dead staining)
A. : Live/Dead cell imaging kit: Cat.# F6627, Thermo Fisher Scientific, USA
2.1.5 Cellular Uptake via ICP-AES
A. Nitric acid: Cat.# 7697-39-2, Sigma-Aldrich, USA B. Chloric acid: Cat.# 7647-01-0, T. J. Baker
2.2 Experimental Instrument and Consumable Materials
2.2.1 Experimental Instrument
A. UV/VIS Spectrophotometer: CARY 100nc, Agilent, USA B. Laminar flow hood
C. Particle Size and Zeta Potential Analyzer: Zetasizer Nano, Malvern, UK D. Incubator: Class-100, Hepa, USA
E. Phase contract optical microscopy: TS-100, Nikon, Japan F. Constant temperature water bath: WB212-B2, KASIN, ROC G. Centrifuge: 5804R, Eppendorf, Germany
H. Orbital shaker: Digisystem Labortory Instruments, INC., ROC I. Analytical balances : AB104-S, Mettler Toledo, USA.
J. Autoclave: Speedy AUTOCLAVE, TOMIN, ROC K. pH meter: Jeno, USA
L. Stirrer/hot plate: model PC-420, Corning, USA M. Oven
N. Hemocytometer
O. Nuclear magnetic resonance (NMR): AVIII-500MHz FT-NMR, Bruker, USA
P. Enzyme-linked immunosorbent assay (ELISA) microplate autoreader: Model EL800, BIO-TEK, USA
Q. Inverted microscope: TS-100, NIKON, Japan
R. Laser power supply: PSU-H-LED, Changchun new industries optoelectronics tech. co. ltd, China
S. 532 nm green single longitudinal mode laser: Class IV laser product, Changchun new industries optoelectronics tech. co. ltd, China T. Fluorescence Microscope: Eclipse 80i microscope ,Nikon, Japan U. ICP-AES: JY 2000-2, Jobin Yvon Horiba, USA
2.2.2 Consumable Materials
A. Syringe filter Unit, 0.22 µm and 0.45 µm: Millipore, USA B. 24-well cell culture plate: Cat.# 142475, Nunc, USA C. 96-well ELISA microplate: Cat.# 269620, Nunc, USA D. 15 mL centrifuge tubes: Cat.# 430829, Corning, USA E. 50 mL centrifuge tubes: Cat.# 430791, Corning, USA
F. 5 mL pipet tips: Cat.# 4487, Corning, USA G. 10 mL pipet tips: Cat.# 4488, Corning, USA
H. Dialysis membrane (molecular weight cutoff: 12-14 kDa) : CelluSep, USA I. 75 mm bottle top filter – 500 mL, Thermo, USA
2.3 Solution Formula
2.3.1 Phosphate Buffered Saline Solution (PBS), pH 7.4.
Besides deionized (DI) water, every 1L of PBS solution contained 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.245 g KH2PO4. The pH of such solution was adjusted to 7.4, sterilized before storage 4°C.
2.3.2 Alpha Minimum Essential Medium (α-MEM) Culture Medium
Culture medium (1L) were made of α-MEM, 3.7 g sodium bicarbonate, 800 µL 2-Mercaptoethanol (0.679 % (v/v)), 10 mL Fungizone, 5 mL Gentamycin, and 10% FBS.
The pH of the prepared solution was adjusted to 7.4 and filtrated with 75 mm bottle top filter.
2.4 Experimental Design
2.4.1 N-[3-(Dimethylamino) propyl] methacrylamide
Among all methods to increase uptake amount of nanogels, the simplest way is using positively surface charged nanogels. In recent years, N-[3-(Dimethylamino) propyl] methacrylamide (NDPMA) were used as a functional monomer in several
researches, which can modify properties of the product polymer. Most of them were used to provide positively surface charge since there’s a tertiary amino terminal group
(Figure 2-1). Bokias et al. synthesized a polymer, in which N-isopropylacrylamide (NIPAAm) was copolymerized with NDPMA, which is positively charged in neutral solution [43]. Chang et al. also used such strategies to improve drug or nucleic acid delivery efficiency [44]. Besides, introducing NDPMA in poly(N-isopropylacrylamide) (pNIPAAm) not only provided positively surface charge, but also increased lower critical solution temperature (LCST) of pNIPAAm. Since NDPMA have already been used and proved to be effective and applicable in numerous researches, we decided to use this compound as major monomer.
2.4.2 Sodium Copper Chlorophyllin
Photothermal molecules are compounds which can convert light (or laser) into local heat effectively. It has been reported that the efficiency of hyperthermia induced by photothermal agents is higher than traditional hyperthermia methods such as
hot-water bath or heated blood perfusion [46]. Thanks to their biocompatibility and biodegradability, certain light-sensitive natural compounds, including porphyrins or chlorophylls, have been investigated for photothermal therapy [47].
Since these molecules contain tricyclic, heterocyclic or porphyrin-like ring -electron system), they are able to convert light into heat and the conversion mechanism can be explained using simple Jablonski diagram (Figure 2-2) [48]. When exposed with light at certain wavelength, conjugated -electrons in these molecules would be excited. This activation results in release of vibrational energy (heat, in most cases), which is what kills the targeted cancer cells.
Specifically, the molecule that has absorbed a light quantum is excited to the first or higher excited states, and dissipated quite rapidly down to the first excited singlet state, which is known as type I mechanism. It is also possible for the singlet state to be transferred into the triplet excited state by intersystem crossing (ISC). There are two different pathways that triplet state involved. One is to transfer energy to molecular oxygen. The other is to transfer electron or hydrogen atom to another molecule [48].
The structure in sodium copper chlorophyllin (SCC), a derivative of chlorophyllin, contains a porphyrin-like ring (Figure 2-3). It should be noted that there are plenty conjugated -electrons in SCC, which gives SCC the potential to generate massive heat through the above mechanisms. Moreover, thanks to its high photothermal
conversion efficiency and low cytotoxicity [49, 50], SCC shows great potential to be used as photothermal agent.
2.4.3 Synthesis and Purification and Characteration of Nanogels
Poly(NDPMA) nanogels (NG), poly(NDPMA-SCC) nanogels (NGC) were synthesized via inverse miniemulsion polymerization in a water-in-oil system. For NG, NDPMA (80 mg, monomer), PEGDA (10 mg, crosslinker) and APS (10 mg, initiator) were dissolved in 3 mL of DI water (a in Figure 2-4). Span80 (1 g, surfactant) were dissolved in 20 mL hexane. Two phases were mixed vigorously in a glass tube with stirrer (b in Figure 2-4). After sealed, the reaction mixture was purged with nitrogen for half an hour to remove dissolved oxygen while being stirred (c in Figure 2-4). Then, the solution was sonicated for 5 minutes to form smaller micelles. The reaction was initiated at 60 oC for 2 hr (d in Figure 2-4). After the reaction (e in Figure 2-4), the solution became turbid comparing to the mixture before reactions.
The reaction procedure of NGC synthesis was almost the same as the description in the above paragraph. The only difference between two procedures was that there was SCC (7 or 21 mg) in DI water in this synthesis. Otherwise were exactly the same.
In purification process, acetone was used to break micelles in the mixture. After adding 5 mL of acetone, the mixture was first centrifuged with 14000 rpm for 20 min.
Another 30 mL of hexane and 5 mL of acetone were added after the removal of the
supernatant. Well mixed and centrifuged at 14000 rpm for another 20 min, the supernatant was removed again and the procedure were repeated 2 times. The solution was diluted with DI water to prevent damage of membrane in syringe filter from organic solvents. Then the solution was filtered through a st
filter sequentially. After washing, the precipitate was further purified by dialysis against water. The final product was lyophilized and stored at 4 oC for further use.
The gold hybrid nanogel (AuNG) used in the cell uptake experiment were synthesized using a stirring process. In 10 mL Au nanoparticle (AuNP) suspension solution (100 ppm), 9 mg of NG were dissolved. The mixture was stirred vigorously with magnetic stirrer for 4 days (g in Figure 2-4). To purify AuNG, the solution containing product naogels was dialyzed against DI water for 2 days and lyophilized, sequentially.
The size distribution of nanogels was analyzed by dynamic light scattering (DLS) with 633 nm wavelength light source. The scattering angle was 173o, and the operations were at room temperature. By measuring its zeta potential, the surface charge of nanogels were determined.
The amount of SCC in NGC was quantified using UV-visible. Briefly, 10 mg of nanogels was dispersed in 1mL DI water and sonicated for 20 min. Such solution was then diluted with DI water to 1 followed by measuring the absorbance at 405
nm wavelength. The unknown SCC contents of NGC were determined by comparing to the values of SCC solutions with known concentrations. The weight percentage of SCC in NGC will be abbreviated as subscript. For example, NGC with a x wt% would be denoted as NGCx.
2.4.4 Thermal Properties and Cytotoxicity of Nanogels
The examination of temperature changes of nanogel solutions required a green laser module source emitting at 532 nm wavelength. Different concentrations of nanogel solutions (1 mL) in eppendorfs were irradiated with laser. The temperature of those solutions was measured by thermocouple and was recorded every minute.
Cytotoxicity of nanogels was determined by MTS assay. L929 cells were seeded 2 x 104 cells/ well in 96-well TCPS plates and incubated under 5% CO2 at 37 oC. After cultured for 24 h, culture mediums were
containing nanogels at various concentrations was added in each well and incubated.
Fresh medium was used as negative control group, while medium containing 0.5 % phenol was used as positive control group. After 2 hr incubation, solution in each well
fresh medium . Three hours later,
the absorption of the solutions in each well were determined using ELISA reader. The optical absorbance was measured at 570 nm (OD570) and 630 nm (OD630). The relative cell viability was calculated by the following equation:
Relative cell viability (% of control)
=(OD570 − OD630 of treated wells)
(OD570 − OD630 of the control) × 100%
2.4.5 Cellular Uptake of Nanogels
105 L929 fibroblasts were seeded in a 3 cm dish for 24 hr and further cultured with the as-prepared nanogels, which were dissolved in fresh cell culture medium to a concentration of 0.1 mg/ mL, for 2 hr (h in Figure 2-4). Then, mediums were removed and the cells were washed with PBS twice to remove free AuNG.
solution in the dish for 5 min to make cells detach from dish bottom. The solution containing L929 cells was transferred into a flask containing 3 mL aqua regia. After 2 hr digestion, the solution was then diluted with DI to a final concentration below 2 % nitric acid. And the gold amount in each samples were detected using ICP-AES (i in Figure 2-4).
To compare the influence of positively surface charged nanogel to those without, the uptake amount of gold for cells incubated with pure AuNP was also determined using the same procedure and equipment.
2.4.6 Viability of L929 cells with nanogels under laser exposure
To observe the cell-killing efficiency of nanogels combining laser irradiation, L929 cells were seeded in 24 well plate, each well contains 105 cells and further incubated for 24 hr. Replaced culture medium with nanogels-containing medium and incubated
for 2 hr. The solutions were removed in each well and washed with PBS to remove free nanogels. After adding 400 mL PBS in each well, L929 cells were irradiated in the well plate with various intensities (1 W, 2 W and 3 W) and times (1min to 5 min). The equipments were setup as the shown in figure 2-1, the area of each well in 24 well plate was 1.9 cm2, and the distance between expander and plate bottom was 5 cm. After exposure, the viability was examined via MTS assay and Live/Dead staining. When operating Live/Dead staining, one dose of Live/Dead® cell imaging kit were diluted in 20 mL PBS and each well to substitute pure PBS. After 20 min incubation, the well plate was observed using fluorescent microscope.
2.4.7 Statistical Analysis
The statistical analysis between different groups were calculated by Student-Neman-Keuls Multiple Comparisons Test (Instat 3.0, Graph Pad Software, USA).
2.4.8 Scheme of this Research
The schemetric illustration of this work were shown in figure 2-2. Briefly, nanogels with or without SCC content were synthesized via inverse miniemulsion polymerization. After purification and lyophilization, part of the produced NG was stirred with AuNP. After purification and lyophilization, the hybrid gold nanogel (AuNG) was produced. AuNG were cultured with L929 to examine the effect of positively surface charged nanogel to cell uptake using ICP-AES. After the determination of cytotoxicity and photothermal property of NG and NGC, the laser-induced cytotoxicity of nanogels were performed.
Figure 2-1. Chemical structure of N-[3-(Dimethylamino) propyl] methacrylamide.
Figure 2-2. Simple Jablonski diagram for explaining the photothermal mechanism [48]
Figure 2-3. Chemical structure of porphyrin ring (A) and sodium copper chlorophyllin (B).
Figure 2-4. Schematic illustration of experiment procedure and setup of this work.
Chapter 3
Positively Charged Nanogels Containing photothermal molecules for Photothermal Therapy
3.1 Characteristics of nanogels
It’s important to quantify the SCC content in our product nanogels. With the use of UV/Vis, we can quantify specific molecules by detecting its absorbance at certain
wavelength. For SCC, the absorbance peak is at 405 nm and 626 nm [51]. Luckily, light with 405 nm wavelength won’t be absorbed by NDPMA molecules. Various
concentrations of SCC solutions were examined to get a calibration curve (Figure 3-1), which can be used to calculate SCC weight percent in each nanogels samples.
Comparing the absorbance of three types of nanogel (NG, NGC) and the calibration curve, three types of nanogels contains 0, 10 and 20 wt% SCC respectively. In the later paragraphs, these nanogels would be denoted as NG, NGC10 and NGC20. From NMR spectrum (Figure 3-2), the area of NDPMA (peak f, 6-H) and SCC (peak g, 2-H) were 5.461:0.053, so the mole ratio is about 33. Convert this result to mass ratio, we get a 10.6 wt% of SCC, which correspond to the result obtained via UV/Vis.
Second, the size of those product gels should be determined. Examined via DLS, size distributions of different nanogels were revealed (Figure 3-3). This result demonstrated that most frequently occurring diameter of NG, AuNG, NGC10 and
NGC20 was at around 250 nm, 190 nm, 220 nm and 255 nm. The size distribution of NG and AuNG is mono-dispersed. The size distribution of NGC10 and NGC20, on the other hand, were broader than that of NG and AuNG, which may result from appearance of SCC. Both the size distribution of NGC10 and NGC20 were not mono-disperse.
Moreover, NGC20 was more chaotic than the size distribution of NGC10. This may suggest that when SCC were involved in reactions, the polymerization process of the nanogels was interfered by SCC.
After stirring AuNP with NG, dialysed and lyophilized, the structure of AuNG were observed using TEM, and from the image (Figure 3-4). It’s interesting that all AuNP were inside NG, and some of NG didn’t contain AuNP. But there’s no free AuNP.
Next, the surface charge of those nanogels should be positive for our use, that’s why we chose NDPMA as monomer. The zeta potential of NG, AuNG, NGC10 and NGC20 were 43.1, 41.2, 49.2 and 38.9 mV respectively, which was positive and corresponding to our expectations.
3.1.1 Photothermal property of the nanogels
The photothermal property of nanogels was determined from the temperature changes of nanogels solution as the irradiated time prolonged. Different kind of nanogels solutions with various concentrations were irradiated with 532 nm green laser, and solution temperature was monitored using a thermal couple and recorded every minute. It appeared that under laser exposure, the temperature of the nanogels solution without SCC content (NG) would not increase whereas that of the solutions containing SCC-incorporated nanogels (NGC10, NGC20) increased significantly. Due to different SCC content, temperature changes in NGC20 group was more than the temperature changes in NGC10 group (Figure 3-5). Also, it is straightforward that the temperature changes increased as the intensity increased. However, when the intensity is higher than 4 W, the temperature of DI water and NG increased slightly after 5 min exposure (Figure 3-6). Thus, in the later laser experiments, the intensities would be limited below 3 W.
3.1.2 Cytotoxicity of nanogels
For biomedical use, cytotoxicity of the materials is crucial. Cytotoxicity of the nanogels were evaluated via MTS assay. Instead of 24 hr incubation, which is commonly used in cytotoxicity test, L929 cells were incubated with nanogels for only 2 hr since particles possess positively surface charge are usually cytotoxic. However, these nanogels can undergo endocytosis process fast, even in such a short time, due to their positively surface charge. After cultured with different nanogels for 2 hr, the
viability in each group was about 80 % Figure
3-7). Moreover, viability in groups with SCC-incorporated nanogels were slightly lower than that in SCC-free nanogel group. Due to positively surface charge, the nanogels possess cytotoxicity, so the viability dropped as the concentration of nanogels increased.
For NGC, NGC20 seems to be less cytotoxic than NGC10, this might result from the different zeta potential. Since NGC20 contained more SCC than NGC10, the surface charge of NGC20 is slightly lower than NGC10.
3.2 Cellular uptake of the nanogels
The uptake of nanogels in L929 cells were examined using ICP-AES. The concentration of Au in two samples, cells incubated with AuNG or AuNP, were 8.5 ppm and 0.1 ppm respectively. Since the volume of two samples were 10 mL, the total
amount of internalized nanogels could be calculated. For each samples there are a total 5 x 105 cells, the average number of AuNG entered a cell is 3579; while that of AuNP is 42. This result proved that positive surface charged nanogels entered easier and faster than neutral ones.
3.3 Laser-induced cytotoxicity of nanogels
To investigate the laser-induced cytotoxicity of nanogels, L929 cell was immersed in mediums with 0.1 mg/mL nanogels concentrations, further cultured for 2 hr and then
irradiated by laser with different intensities and time duration. The MTS results shown that when cells were cultured with NG, viability wouldn’t decrease as exposure time
irradiated by laser with different intensities and time duration. The MTS results shown that when cells were cultured with NG, viability wouldn’t decrease as exposure time