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Photo-crosslinked natural polyelectrolyte multilayer capsules for drug delivery
Accepted Manuscript Title: Photo-crosslinked natural polyelectrolyte multilayer capsules for drug delivery Author: Narisu Hu Johannes Frueh Ce Zheng Bin Zhang Qiang He PII: DOI: Reference:
S0927-7757(15)30033-9 http://dx.doi.org/doi:10.1016/j.colsurfa.2015.06.014 COLSUA 19957
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-3-2015 5-6-2015 7-6-2015
Please cite this article as: Narisu Hu, Johannes Frueh, Ce Zheng, Bin Zhang, Qiang He, Photo-crosslinked natural polyelectrolyte multilayer capsules for drug delivery, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2015.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Photo-crosslinked Natural Polyelectrolyte Multilayer Capsules for Drug Delivery
Narisu Hua,b, Johannes Fruehb, Ce Zhengc, Bin Zhanga,d,e*, Qiang Heb
a
Oral Implant Center, Second Affiliated Hospital of Harbin Medical University, Harbin
150086, China b
Key Laboratory of Microsystems and Microstructures Manufacturing, Micro/Nano
Technology Research Centre, Harbin Institute of Technology, Yikuang Street 2, Harbin 150080, China c d
Medical Affairs Department, Harbin Medical University, Harbin 150001, China Sino-Russian Institute of Hard Tissue Development and Regeneration, Second
Affiliated Hospital of Harbin Medical University, Harbin 150086, China e
Heilongjiang Academy of Medical Sciences, Harbin 150086, China
Corresponding Author: Prof. Bin Zhang Oral Implant Center Second Affiliated Hospital of Harbin Medical University Harbin 150086, China E-mail: [email protected] Highlights
Chitosan and Alginate PEMs can be crosslinked using 4-vinyl-benzyl chloride Diffusion of Fluorescein isothiocyanate through the PEM is decreased by factor 3.8 Diffusion of model drugs like doxorubicin hydrochloride is decreased by factor 2.5 PEM crosslinking using 4-vinyl-benzyl chloride is done by a 2 step reaction
The used PEM is biodegradable and not poisonous Abstract
Polyelectrolyte multilayer (PEM) capsules have been widely investigated in drug delivery systems in past two decades, but still remain challenging for their uncontrolled release properties and kinetics. One way to control diffusion-based drug release is to crosslink the PEMs so that the permeability of capsules can be decreased and thus, the encapsulated drugs could be released with respect to the changes of chemical, physical or biological conditions. The photo-crosslinking method is approximately 16 times faster than the normal chemical methods. We hereby present a fast photo-crosslinking method of chitosan-alginate PEM capsules, which allows effectively decreasing the diffusion of the encapsulated components across the PEM walls of capsules. The experimental results show that photocrosslinked capsules exhibit a significantly lower release rate of anticancer drug, doxorubicine compared to non-crosslinked capsules, demonstrating the usability of this method in controlled drug delivery and biomedical applications. Key words: layer-by-layer, photo-crosslinking, capsule, drug delivery, biodegradability
1. Introduction Polyelectrolyte multilayer films (PEM) produced by the layer-by-layer (LbL) assembly technique[1,2] find applications in many fields ranging from cell culture supports[3], tuning optical properties of interfaces [4] to antibiofouling modification [5,6]. The reason for the utilization of PEMs in such a diversity of fields is the possibility to structure their thickness with nanometer precision and the ability to tune their internal properties.[7] These films can contain a large variety of internal structures, depending
on the used ionic strength[8,9] during the deposition process or the type of embedded materials [10]. The LbL-assembled PEMs are commonly formed via electrostatic interaction [1], hydrogen bonding [2] or covalent bonding [11]. Also, the PEMs via electrostatic LbL-assembly can be further chemically crosslinked PEM films to enhance the stability or decrease the permeability.[12] Basically, the crosslinking was mainly performed by heating the PEM films or using chemical crosslinkers, which causes the reduction of the amount of incorporated entities or longer crosslinking time.[13-19] More recently the crosslinking with photo-active molecules was introduced, which allows a drastically decreased crosslinking time, whereby defined patterns for tuning optical properties or an increased film rigidity for cell adsorption was created.[20,21] One of the great advantages of this method is to avoid poisonous chemicals like glutaraldehyde.[15] LbL-assembled PEM capsules[22] have gained significant interests as controlled drug encapsulation and release systems. The initial studies in this area focused on the use of synthetic polyelectrolytes for the build-up of multilayer films. Now it has been expanded to utilise a variety of different materials including inorganic nanoparticles, dyes, dendrimers, peptides, DNA, and enzymes as assembly components. However, how to effectively trigger the release of the encapsulated components inside capsules with respect to biological stimuli such as enzymes, still remains challenging.[23] In this study, we report biodegradable, photocrosslinkable PEM capsules as drug delivery systems. The capsules are made from biodegradable chitosan (CHI) and alginate (ALG) to show the possibility of crosslinked PEM
films for tunable diffusion
through the capsule walls. The photocrosslinkable unit used was 4-vinylbenzene which was chemically linked to the ALG. Manufacturing PEM capsules with and without
crosslinking allowed for measurement of altered dye and drug diffusion through the membrane due to the crosslinking. Results hint structural changes not only in the mechanical properties[24,25] of the PEM but also in voids[26,27] and channel structures.
2. Materials and Methods 2.1 Materials: The used polyelectrolytes chitosan (CHI, medium molecular weight), and Alginic acid sodium salt (ALG) (extracted from brown Algae), 4-vinyl-benzyl chloride (VB, technical grade, purity >90 %) were bought from Sigma (St Louis, USA). The linking of the VB to ALG was achieved through an ester-bond formation according to the well-established procedure for hydrophobic group linkage to acetate groups of ALG developed by Duval.[21,28-31] 2.2 Synthesis of VB-modified ALG Briefly, the ALG was first transformed into alginic acid by percolating it through a cationic exchange resin (Amberlite IR-120 H+ form, Sigma, St. Luis, USA). The alginic acid was then neutralized with tetrabutylammonium hydroxide (C4H5)4N+OH- (Beijing Chemical works, China). After freeze drying the PE, it was dried in vacuum at 40 °C for 1 day. The alginic acid was dissolved in dried DMSO at 40 °C. Then the VB at a concentration of 0.0183 g/mL was added at a weight ratio equal to 45 % of the ALG monomer-mol% to the solution. Then the solution was stirred by a magnetic stirrer for 2 days. The alginic acid was converted back to the sodium salt by adding a 10% (w/w) NaCl solution. Then 900mL of acetone was added and the precipitate was filtered, washed with 400mL of acetone and dried. The resulting VB-ALG was purified by
dialysis against ultra-pure water for 1 week, whereby the ultra-pure water was exchanged daily. The VB-ALG was subsequently dried. Scheme 1 shows the resulting molecular structure of the ALG-VB. 2.3 LbL assembly of VB-ALG/CHI multilayer: The PEM films were produced by the LbL dipping procedure[1,2] by immersing piranha cleaned quartz slides (50% H2SO4 (98%, purity p.a., Chemical Reagents Tianjin, China) with 50% H2O2 (30% stabilized Chemical Reagents Tianjin, China, purity p. a.) for 50 minutes) alternatively into PE solutions of either CHI or ALG (0.5g/L at a ionic strength of 0.15M NaCl (Chemical Reagents Tianjin, China). The pH of the PE solution was adjusted to 7.4 with 0.1M NaOH (Chemical Reagents, Tianjin, China), the used pH electrode was a Sartorius PB-10 (Sartorius, Goettingen, Germany). The dipping time for each PE solution was 8 minutes, whereby each PE solution dipping step was followed by 3 washing steps in ultra-pure water (30 seconds for each washing step) (resistance >18 MΩcm-1, Elga Labwater, Beijing, China). The crosslinking of the CHI/ALG-VB films was performed by irradiating the PEM with 254 nm UV light produced by a ZF-7A UV lamp purchased from Shanghai Baoshang Gucun, Shanghai, China. The PEM capsules were irradiated before the core was dissolved, which prevented a sideward contraction of the capsule to ensure comparability of the diffusion through the PEM between crosslinked and non crosslinked samples. It is noted that for comparison experiments also a crosslinking after core dissolution was performed. 2.4 Preparation of VB-ALG/CHI capsules: The PEM capsules were produced by coating silica particles (20 µm diameter, Nano-Micro, Hangzhou, China) with PE concentrations of 1 g/L and subsequent separation of the particles and solution by centrifugation of the particles according to
reference [22]. The particles were washed with ultra-pure water 3 times after every PE adsorption step. After depositing the PEM onto the particles, the template core was dissolved with HF (Chemical Reagents, Tianjin, China). 2.5 Encapsulation of FITC-dextran and DOX Fluorescein isothiocyanate (FITC)-labeled dextran and doxorubicin hydrochloride (DOX) were purchased from Sigma (St. Louis USA) and used as cargo for the capsules, whereby the loading of the capsules can be achieved by simply co- incubating the capsules with DOX or FITC-dextran solutions for 24 hours. The used DOX and FITC-dextran concentrations were 0.25 mg/mL and 0.2 mg/mL. 2.6 Characterization Extinction and absorption spectra were recorded with a Hitachi U4100 (Tokyo, Japan) UV-vis-near-infrared spectrophotometer. Fluorescence images were obtained using a Leica TCS SP5 II confocal laser scanning microscope (CLSM). The excitation wavelength was 488 nm for the excitation of FITC. The zeta potential measurements were performed using a ZetaPALS (Brookhaven Instrument Corporation, Holtsville, NY, USA) Zeta sizer. 2.7 Degradation of the PEM capsules The as-prepared capsules were exposed to a pancreatin solution (5 U/mL) in phosphate buffer solution (PBS) at pH 7.4 at room temperature overnight. As a control, the capsules without enzyme treatment were incubated in plain PBS at pH 7.4 at room temperature. The samples were then centrifuged, removed from the incubation solution and washed three times for SEM characterization.
3. Results and discussion
3.1 Linking of the VB The successful linking of VB to the ALG was confirmed by measuring UV spectra of the solution (the labelling degree was estimated with the concentration of the PE, as well as the known extinction coefficient of the vinyl group, 18390L (mol×cm)) as well as PEM
films. The VB compound itself would not be able to form films based on
electrostatic overcompensation, since it bears no charge. Following the absorption of the VB peak at 252 nm, one can see a linear increase with the PEM layer number in Fig. 1. The extinction increases linearly, whereby the absorption increases exponentially as seen in Fig. 2a, b. Although the absorption is known to increase exponentially for matter, the absorption of a monolayer or bilayer is negligible, as can be seen for the low starting values of the absorption peak in Fig. 2. These values rise quickly with increasing layer numbers, due to the strong inter-diffusion of the PEs between the new and old layers, which is common for weak PE based PEM films.[7,32] It should be pointed out that the formed VB-ALG/CHI multilayer films are estimated to have a crosslinking degree of about 34±3 %, which leads not only to a strong coiling, but also to a decreased charge density of the PEs. Interestingly, the formed VB-ALG/CHI multilayers have a higher crosslinking density compared to previously reported VB-crosslinked polyacrylic acid.[21,31] The fact that our ALG-VB is able to form films with such a high labeling degree shows, that this kind of PE are not only forming films due to electrostatic but also due to hydrophobic and dipole interaction. The result film is soft, with strong inter-digitation and exponential growth.[33] Note that the crosslinking behavior is different compared to linear growing azo-benzene based photocrosslinkable films since our film crosslinking is much faster and has higher crosslinking degree.[34]
An interesting fact is, that despite the low charge degree this type of PEM film is still able to reverse the charge degree of the surface as can be seen in Fig. 3. This effect proves, that despite the large VB labeling degree and additional shielding from 0.15 M counterion strength the PEs are able to cause a surface charge overcompensation. Shining UV light onto these CHI/ALG-VB modified PEM films in flat conditions allows us to photo-crosslink the PEM comparable to reference [28]. Fig. 4 a) shows the decrease of the absorption peak at 252 nm, while b) shows the decrease in extinction and relative absorption with exponential decay fits over time. Although the relative absorption decreases like in case of reference [28] double exponentially, we argue their explanation for the crosslinking only occurring between the VB groups. This is because hydrophobic groups form usually intra and not intermolecular dimers or micro-phase separations.[35,36] An intramolecular crosslinking would prevent a decoiling[35] and shear force based re-alignment[24,37] of the PE upon elongation or introduction of mechanical stress, and the explanation goes well with the observed shrinking[28] for these films. The degree of swelling and decreased swelling for these films in water[21] is however not in line with this observation, since the water could still fill the voids between the PE and swell the whole cation, while only the anion swelling would be hindered. Therefore the swelling would be decreased by only 1/3 according to the model in reference [28] which facilitates a single exponential decrease in the absorption peak. Since the absorption peak decreases double exponentially and the decrease in swelling is 2/3, we propose a different model for the photo-crosslinking. The fast component is the VB-VB crosslinking, which is indeed quite fast and mentioned in reference [28]. The second component causes the strong crosslinking, for reactions, which are usually considered “side reactions” in radical polymerization. The
photo-induced radicals of the former vinyl group might rip out hydrogen atoms of the backbone of the CHI and subsequently crosslink with the resulting carbon radical, causing a linkage between the two chains. The fact, that these types of films also contain water, should result also in the formation of alcohol groups as well as a variety of intra- instead of intermolecular interlinks, explaining the observed lower degree of crosslinking and mechanical strength reported in reference [28]. Due to the fact that polysugars like PLL, ALG and CHI do contain alcohol groups already makes the detection of newly formed alcohol groups difficult. One could detect the change in numbers with sensitive techniques like quantitative ATR as reported for PEM in reference [24], which is however not available in our lab. An alternative for the test of our theory might also be performed using PAA based films; however the low thickness of these films might require ATR setups as well. 3.2 Structure of the produced capsules Comparing SEM images of the PEM capsules with and without UV treatment, which were incubated for 25 hours in different pH media (see Fig. 5) one can observe an increased stability (PEM stays thicker) for crosslinked PEM in high and low pH values. In addition the morphological changes to a slightly smoother PEM (compare Fig. 5 c and d). The inner structures of PEM
films suffer not only a contraction, but a structural
change upon crosslinking as well. Estimating a simple contraction of the PEM, with a constant pore diameter wi the PEM, one would expect an increased diffusion through the PEM. Interestingly the contrary is the case, the diffusion decreased by factor 3.8±0.25 for simple small dye molecules like FITC as shown in Fig. 6. This decrease proves that small pores and voids are closed upon crosslinking, which might hint a
contraction of our PEM film, whereby the voids and pores are closed upon contraction. This would however only be possible if the voids would not lead in a straight way through the PEM which has an approximate thickness of 1 µm. The pores are usually in the nano-range and were until now only observed in the special case of strongly swollen PEM of low pH or extremely high salt concentration, where the PEM was already in state of being dissolved.[31,38] Alternatively a multi-dimensional contraction of the pores is also possible, however unlikely, since the core was dissolved after the crosslinking, so the contraction of the sides was limited, whereby a complete internal re-arrangement cannot be ruled out completely. Preliminary tests of the core being dissolved before crosslinking however showed no shrinking, which is probably due to the PEM being crosslinked and hardening faster than the PEM diffusion on large length scales. The fact that PEM hydrophobic moieties are usually aligned along the surface plane and are not isotropically oriented supports our theory that the pores are not leading in a straight line through the PEM but in an indirect way, resulting in an easy to shrink or close path[37]. The remaining question, which could not be solved, is weather just a large amount of pores were closed while direct ones staid open, or all channels simply decreased in diameter. Our pore closing theory is supported by SEM images, showing a smoother surface after crosslinking (see Fig. 5). 3.3 Drug delivery properties of the produced capsules Drug delivery experiments using these capsules showed that the crosslinked capsules are releasing a model drug (DOX) much slower compared to non crosslinked capsules as can be seen in Fig. 7. It is worth noting, that an asymptotic fit failed for this kind of sample and therefore an allomeric fit was used. Using an allometric fit (
, with x
being the time, Y being the released DOX in % and a and b being constants) to
determine the steepness of the release rate, one gets for non-crosslinked capsules a pre-exponent of 60 for a, while it is only 40 for crosslinked capsules. This difference in 50% in the pre-exponential factor is due to the increased diffusion time. Interestingly the exponential constant for the crosslinked samples is much higher than for the non-crosslinked samples (0.22 versus 0.08). This observation is owed by the fact that the non-crosslinked capsules released their DOX already before they could be measured in the UV spectrometer. Therefore these samples have a higher offset and a low increase since the plateau is already reached in the first half hour. The crosslinked samples on the contrary only lost 50% of their cargo and show therefore a stronger increase after the first 30 minutes. Comparing the increase in release after the first 30 minutes, we observe a difference in diffusion by factor 2.5 between crosslinked and non-crosslinked capsules, which are closer to the FITC diffusion values measured by FRAP in Fig 6. The fact, that the release rate does not reach 100% in our experiments is due to the fact that in the first washing steps a considerable amount (20-30%) of DOX is already released and removed. Although the drug release rate is in our case much faster and less easy to control as in case of pH triggered release[39] it provides a novel path to deliver drugs. We would like to point out, that our approach is especially interesting for cases, where the drug delivery is necessary, but no clear pH gradient is present. In such cases a diffusion dependent release might be better than a delivery system requiring a alkaline pH[39], which is present in parts of the human or cellular stomach systems. To investigate if our crosslinked capsules show a pH dependent change in diffusion coefficient (e.g. if smaller versions could be eaten in form of a therapy) we incubated crosslinked and non-crosslinked capsules into a FITC containing media at pH 3, 7, and
11. As shown in Fig. 9 the crosslinked capsules are pH responsive, whereby low pH causes an increased diffusion through the capsule wall, probably due to acid based disassembly of the PEs. In case of pH 7 the diffusion coefficient is lowest, while for pH 11 a slightly increased diffusion coefficient is observed. For non-crosslinked capsules the diffusion coefficient is high for all pH ranges. This finding is surprising and allows for utilization of our drug carriers in case of endosome or lysosome based drug release systems in the future.[40] To investigate the biodegradability of our novel photocrosslinkable drug delivery systems, we incubated crosslinked and non-crosslinked capsules in pancreatin solution and investigated the structural change of the capsules after one night. As can be seen in Fig. 10, both the crosslinked and non-crosslinked capsules were significantly degraded. The crosslinking reduced the degradation degree to some extent, but the biodegradability was preserved. The fact that a human body the temperature is 37 and not 20 °C, will allow a significantly higher degree of biodegradation than in this study. The capsules were neither in crosslinked nor in pristine form poisonous to HELA cells (Fig. 11 a). Upon embedding DOX into the capsules, the viability of the HELA cells could be significantly decreased depending on the amount of embedded DOX (Fig. 11 b). This proves the usability of the proposed drug delivery system.
4. Conclusion We successfully crosslinked PEM films comprising out of ALG and CHI. The crosslinking mechanism exhibits 2 components, which we suspect to stem from reactions between the vinyl groups and side reactions, from crosslinking with the backbone of the counter PE, since PE of the same kind are shielded from each other due
to charge repulsion. We also show for the first time, that it is possible to produce photo-crosslinked PEM capsules. These crosslinked PEM capsules show a decreased diffusion of dyes through their membrane compared to non crosslinked capsules of the same diameter and same composition. Drug delivery experiments showed a clear decrease in
the diffusion rate and release of DOX compared to non-crosslinked
counterparts. This allows a controlled release over longer time periods for example overnight. In case of placing recognition sites onto the capsules to detect tumor sites, the decreased release rate allows the capsules to stay a longer time in the body until they reach the tumor site due to the blood flow. The proposed system can easily be used to produce smaller capsules by simply using smaller templates. In addition the used PEM system is biodegradable yielding no poisonous substances.
Acknowledgements This work was financially supported by Natural Science Foundation of China (Grant No. 81170960),
Startup
Grant
of HIT,
China
Postdoctoral
Science
Foundation
(2013M531019) and Foundation of the Department of Science and Technology of Heilongjiang Province (Grant No. GC12C303-2).
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Figure captions:
Scheme 1. Structure of vinylbenzene (VB) chemically linked to Alginate
Fig. 1. UV absorption spectra of ALG-VB/CHI PEM films. Films without VB do not show the absorption peak at 252 nm. Fig. 2. Comparison of a) Extinction and b) inverse absorption of the VB absorption peak at 252 nm. The PEM grows single exponentially which is typical for this type of PEM films. Layers of the X axis in a) and b) correspond to mono layers of single PE. Lines in a) and b) are linear and exponential fits. Fig. 3. Zeta potential measurement of 1 µm capsules for several deposition cycles. Fig. 4. a) Absorption spectra of the 252 nm absorption peak of the vinyl group in ALG-VB upon UV irradiation. After 45 minutes of irradiation this peak is nearly totally gone; b) Intensity of the 252 nm absorption peak in extinction with a single exponential decay fit, whereby the inset shows the relative absorption with single and double exponential decay fits. Fig. 5. The SEM images of the capsule with (A, C, and E) and without UV irradiation (B, D, and F) in pH 3 (A and B), pH 7 (C and D), and pH 11 (E and F). Fig. 6. Comparison of the fluorescence recovery after photo-bleaching of FITC for a)-d) not crosslinked and e)-h) photo-crosslinked PEM capsules. The non crosslinked capsules recover the fluorescence faster as a comparison of the intensity versus time plots for non-crosslinked i) and crosslinked j) PEM capsules shows. Fig. 7. Drug (DOX) release properties of crosslinked and not crosslinked PEM capsules. a) Normalized release rate of PEM capsules, b) release rate versus time. The used allometric fit in a) and the exponential decay fit in b) show a clear discrepancies between crosslinked and non crosslinked capsules. The release in percent can be seen in Fig. 8. Fig. 8. Drug (DOX) release properties of crosslinked and not crosslinked PEM capsules.
The used allometric fit shows clear discrepancies between crosslinked and non crosslinked capsules. Fig. 9. The CLSM images of the cross linked capsules (A, B, and C) and non-cross linked capsule (D, E, and F) under pH 3 (A and D), pH 7 (B and E), and pH 11 (C and F). Scale bar 20 µm. Diffusion constant of capsule wall different for crosslinked and non-crosslinked ones as well as for different pH. Fig. 10. The SEM images of the capsule without treatment (A), under enzyme treatment (B), under UV irradiation (UV irradiation), and under the enzyme treatment and UV irradiation (D). Images taken after incubating the capsules for 1 night in pancreatin solution at room temperature. Fig. 11. Cells viability essay (MTT) of cells being incubated with (A) not crosslinked and crosslinked capsules. (B) Crosslinked capsules loading different concentration of DOX. As can be seen the drug can be successfully be delivered to HELA cells. Figures and Schemes H
H
H OH
HO COONa
OH
H H
OH
O H OH
O
H H OH
O
C O
O
Scheme 1.
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.
Fig. 8.
Fig. 9.
Fig. 10.
Fig. 11.
S0927-7757(15)30033-9 http://dx.doi.org/doi:10.1016/j.colsurfa.2015.06.014 COLSUA 19957
To appear in:
Colloids and Surfaces A: Physicochem. Eng. Aspects
Received date: Revised date: Accepted date:
13-3-2015 5-6-2015 7-6-2015
Please cite this article as: Narisu Hu, Johannes Frueh, Ce Zheng, Bin Zhang, Qiang He, Photo-crosslinked natural polyelectrolyte multilayer capsules for drug delivery, Colloids and Surfaces A: Physicochemical and Engineering Aspects http://dx.doi.org/10.1016/j.colsurfa.2015.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Photo-crosslinked Natural Polyelectrolyte Multilayer Capsules for Drug Delivery
Narisu Hua,b, Johannes Fruehb, Ce Zhengc, Bin Zhanga,d,e*, Qiang Heb
a
Oral Implant Center, Second Affiliated Hospital of Harbin Medical University, Harbin
150086, China b
Key Laboratory of Microsystems and Microstructures Manufacturing, Micro/Nano
Technology Research Centre, Harbin Institute of Technology, Yikuang Street 2, Harbin 150080, China c d
Medical Affairs Department, Harbin Medical University, Harbin 150001, China Sino-Russian Institute of Hard Tissue Development and Regeneration, Second
Affiliated Hospital of Harbin Medical University, Harbin 150086, China e
Heilongjiang Academy of Medical Sciences, Harbin 150086, China
Corresponding Author: Prof. Bin Zhang Oral Implant Center Second Affiliated Hospital of Harbin Medical University Harbin 150086, China E-mail: [email protected] Highlights
Chitosan and Alginate PEMs can be crosslinked using 4-vinyl-benzyl chloride Diffusion of Fluorescein isothiocyanate through the PEM is decreased by factor 3.8 Diffusion of model drugs like doxorubicin hydrochloride is decreased by factor 2.5 PEM crosslinking using 4-vinyl-benzyl chloride is done by a 2 step reaction
The used PEM is biodegradable and not poisonous Abstract
Polyelectrolyte multilayer (PEM) capsules have been widely investigated in drug delivery systems in past two decades, but still remain challenging for their uncontrolled release properties and kinetics. One way to control diffusion-based drug release is to crosslink the PEMs so that the permeability of capsules can be decreased and thus, the encapsulated drugs could be released with respect to the changes of chemical, physical or biological conditions. The photo-crosslinking method is approximately 16 times faster than the normal chemical methods. We hereby present a fast photo-crosslinking method of chitosan-alginate PEM capsules, which allows effectively decreasing the diffusion of the encapsulated components across the PEM walls of capsules. The experimental results show that photocrosslinked capsules exhibit a significantly lower release rate of anticancer drug, doxorubicine compared to non-crosslinked capsules, demonstrating the usability of this method in controlled drug delivery and biomedical applications. Key words: layer-by-layer, photo-crosslinking, capsule, drug delivery, biodegradability
1. Introduction Polyelectrolyte multilayer films (PEM) produced by the layer-by-layer (LbL) assembly technique[1,2] find applications in many fields ranging from cell culture supports[3], tuning optical properties of interfaces [4] to antibiofouling modification [5,6]. The reason for the utilization of PEMs in such a diversity of fields is the possibility to structure their thickness with nanometer precision and the ability to tune their internal properties.[7] These films can contain a large variety of internal structures, depending
on the used ionic strength[8,9] during the deposition process or the type of embedded materials [10]. The LbL-assembled PEMs are commonly formed via electrostatic interaction [1], hydrogen bonding [2] or covalent bonding [11]. Also, the PEMs via electrostatic LbL-assembly can be further chemically crosslinked PEM films to enhance the stability or decrease the permeability.[12] Basically, the crosslinking was mainly performed by heating the PEM films or using chemical crosslinkers, which causes the reduction of the amount of incorporated entities or longer crosslinking time.[13-19] More recently the crosslinking with photo-active molecules was introduced, which allows a drastically decreased crosslinking time, whereby defined patterns for tuning optical properties or an increased film rigidity for cell adsorption was created.[20,21] One of the great advantages of this method is to avoid poisonous chemicals like glutaraldehyde.[15] LbL-assembled PEM capsules[22] have gained significant interests as controlled drug encapsulation and release systems. The initial studies in this area focused on the use of synthetic polyelectrolytes for the build-up of multilayer films. Now it has been expanded to utilise a variety of different materials including inorganic nanoparticles, dyes, dendrimers, peptides, DNA, and enzymes as assembly components. However, how to effectively trigger the release of the encapsulated components inside capsules with respect to biological stimuli such as enzymes, still remains challenging.[23] In this study, we report biodegradable, photocrosslinkable PEM capsules as drug delivery systems. The capsules are made from biodegradable chitosan (CHI) and alginate (ALG) to show the possibility of crosslinked PEM
films for tunable diffusion
through the capsule walls. The photocrosslinkable unit used was 4-vinylbenzene which was chemically linked to the ALG. Manufacturing PEM capsules with and without
crosslinking allowed for measurement of altered dye and drug diffusion through the membrane due to the crosslinking. Results hint structural changes not only in the mechanical properties[24,25] of the PEM but also in voids[26,27] and channel structures.
2. Materials and Methods 2.1 Materials: The used polyelectrolytes chitosan (CHI, medium molecular weight), and Alginic acid sodium salt (ALG) (extracted from brown Algae), 4-vinyl-benzyl chloride (VB, technical grade, purity >90 %) were bought from Sigma (St Louis, USA). The linking of the VB to ALG was achieved through an ester-bond formation according to the well-established procedure for hydrophobic group linkage to acetate groups of ALG developed by Duval.[21,28-31] 2.2 Synthesis of VB-modified ALG Briefly, the ALG was first transformed into alginic acid by percolating it through a cationic exchange resin (Amberlite IR-120 H+ form, Sigma, St. Luis, USA). The alginic acid was then neutralized with tetrabutylammonium hydroxide (C4H5)4N+OH- (Beijing Chemical works, China). After freeze drying the PE, it was dried in vacuum at 40 °C for 1 day. The alginic acid was dissolved in dried DMSO at 40 °C. Then the VB at a concentration of 0.0183 g/mL was added at a weight ratio equal to 45 % of the ALG monomer-mol% to the solution. Then the solution was stirred by a magnetic stirrer for 2 days. The alginic acid was converted back to the sodium salt by adding a 10% (w/w) NaCl solution. Then 900mL of acetone was added and the precipitate was filtered, washed with 400mL of acetone and dried. The resulting VB-ALG was purified by
dialysis against ultra-pure water for 1 week, whereby the ultra-pure water was exchanged daily. The VB-ALG was subsequently dried. Scheme 1 shows the resulting molecular structure of the ALG-VB. 2.3 LbL assembly of VB-ALG/CHI multilayer: The PEM films were produced by the LbL dipping procedure[1,2] by immersing piranha cleaned quartz slides (50% H2SO4 (98%, purity p.a., Chemical Reagents Tianjin, China) with 50% H2O2 (30% stabilized Chemical Reagents Tianjin, China, purity p. a.) for 50 minutes) alternatively into PE solutions of either CHI or ALG (0.5g/L at a ionic strength of 0.15M NaCl (Chemical Reagents Tianjin, China). The pH of the PE solution was adjusted to 7.4 with 0.1M NaOH (Chemical Reagents, Tianjin, China), the used pH electrode was a Sartorius PB-10 (Sartorius, Goettingen, Germany). The dipping time for each PE solution was 8 minutes, whereby each PE solution dipping step was followed by 3 washing steps in ultra-pure water (30 seconds for each washing step) (resistance >18 MΩcm-1, Elga Labwater, Beijing, China). The crosslinking of the CHI/ALG-VB films was performed by irradiating the PEM with 254 nm UV light produced by a ZF-7A UV lamp purchased from Shanghai Baoshang Gucun, Shanghai, China. The PEM capsules were irradiated before the core was dissolved, which prevented a sideward contraction of the capsule to ensure comparability of the diffusion through the PEM between crosslinked and non crosslinked samples. It is noted that for comparison experiments also a crosslinking after core dissolution was performed. 2.4 Preparation of VB-ALG/CHI capsules: The PEM capsules were produced by coating silica particles (20 µm diameter, Nano-Micro, Hangzhou, China) with PE concentrations of 1 g/L and subsequent separation of the particles and solution by centrifugation of the particles according to
reference [22]. The particles were washed with ultra-pure water 3 times after every PE adsorption step. After depositing the PEM onto the particles, the template core was dissolved with HF (Chemical Reagents, Tianjin, China). 2.5 Encapsulation of FITC-dextran and DOX Fluorescein isothiocyanate (FITC)-labeled dextran and doxorubicin hydrochloride (DOX) were purchased from Sigma (St. Louis USA) and used as cargo for the capsules, whereby the loading of the capsules can be achieved by simply co- incubating the capsules with DOX or FITC-dextran solutions for 24 hours. The used DOX and FITC-dextran concentrations were 0.25 mg/mL and 0.2 mg/mL. 2.6 Characterization Extinction and absorption spectra were recorded with a Hitachi U4100 (Tokyo, Japan) UV-vis-near-infrared spectrophotometer. Fluorescence images were obtained using a Leica TCS SP5 II confocal laser scanning microscope (CLSM). The excitation wavelength was 488 nm for the excitation of FITC. The zeta potential measurements were performed using a ZetaPALS (Brookhaven Instrument Corporation, Holtsville, NY, USA) Zeta sizer. 2.7 Degradation of the PEM capsules The as-prepared capsules were exposed to a pancreatin solution (5 U/mL) in phosphate buffer solution (PBS) at pH 7.4 at room temperature overnight. As a control, the capsules without enzyme treatment were incubated in plain PBS at pH 7.4 at room temperature. The samples were then centrifuged, removed from the incubation solution and washed three times for SEM characterization.
3. Results and discussion
3.1 Linking of the VB The successful linking of VB to the ALG was confirmed by measuring UV spectra of the solution (the labelling degree was estimated with the concentration of the PE, as well as the known extinction coefficient of the vinyl group, 18390L (mol×cm)) as well as PEM
films. The VB compound itself would not be able to form films based on
electrostatic overcompensation, since it bears no charge. Following the absorption of the VB peak at 252 nm, one can see a linear increase with the PEM layer number in Fig. 1. The extinction increases linearly, whereby the absorption increases exponentially as seen in Fig. 2a, b. Although the absorption is known to increase exponentially for matter, the absorption of a monolayer or bilayer is negligible, as can be seen for the low starting values of the absorption peak in Fig. 2. These values rise quickly with increasing layer numbers, due to the strong inter-diffusion of the PEs between the new and old layers, which is common for weak PE based PEM films.[7,32] It should be pointed out that the formed VB-ALG/CHI multilayer films are estimated to have a crosslinking degree of about 34±3 %, which leads not only to a strong coiling, but also to a decreased charge density of the PEs. Interestingly, the formed VB-ALG/CHI multilayers have a higher crosslinking density compared to previously reported VB-crosslinked polyacrylic acid.[21,31] The fact that our ALG-VB is able to form films with such a high labeling degree shows, that this kind of PE are not only forming films due to electrostatic but also due to hydrophobic and dipole interaction. The result film is soft, with strong inter-digitation and exponential growth.[33] Note that the crosslinking behavior is different compared to linear growing azo-benzene based photocrosslinkable films since our film crosslinking is much faster and has higher crosslinking degree.[34]
An interesting fact is, that despite the low charge degree this type of PEM film is still able to reverse the charge degree of the surface as can be seen in Fig. 3. This effect proves, that despite the large VB labeling degree and additional shielding from 0.15 M counterion strength the PEs are able to cause a surface charge overcompensation. Shining UV light onto these CHI/ALG-VB modified PEM films in flat conditions allows us to photo-crosslink the PEM comparable to reference [28]. Fig. 4 a) shows the decrease of the absorption peak at 252 nm, while b) shows the decrease in extinction and relative absorption with exponential decay fits over time. Although the relative absorption decreases like in case of reference [28] double exponentially, we argue their explanation for the crosslinking only occurring between the VB groups. This is because hydrophobic groups form usually intra and not intermolecular dimers or micro-phase separations.[35,36] An intramolecular crosslinking would prevent a decoiling[35] and shear force based re-alignment[24,37] of the PE upon elongation or introduction of mechanical stress, and the explanation goes well with the observed shrinking[28] for these films. The degree of swelling and decreased swelling for these films in water[21] is however not in line with this observation, since the water could still fill the voids between the PE and swell the whole cation, while only the anion swelling would be hindered. Therefore the swelling would be decreased by only 1/3 according to the model in reference [28] which facilitates a single exponential decrease in the absorption peak. Since the absorption peak decreases double exponentially and the decrease in swelling is 2/3, we propose a different model for the photo-crosslinking. The fast component is the VB-VB crosslinking, which is indeed quite fast and mentioned in reference [28]. The second component causes the strong crosslinking, for reactions, which are usually considered “side reactions” in radical polymerization. The
photo-induced radicals of the former vinyl group might rip out hydrogen atoms of the backbone of the CHI and subsequently crosslink with the resulting carbon radical, causing a linkage between the two chains. The fact, that these types of films also contain water, should result also in the formation of alcohol groups as well as a variety of intra- instead of intermolecular interlinks, explaining the observed lower degree of crosslinking and mechanical strength reported in reference [28]. Due to the fact that polysugars like PLL, ALG and CHI do contain alcohol groups already makes the detection of newly formed alcohol groups difficult. One could detect the change in numbers with sensitive techniques like quantitative ATR as reported for PEM in reference [24], which is however not available in our lab. An alternative for the test of our theory might also be performed using PAA based films; however the low thickness of these films might require ATR setups as well. 3.2 Structure of the produced capsules Comparing SEM images of the PEM capsules with and without UV treatment, which were incubated for 25 hours in different pH media (see Fig. 5) one can observe an increased stability (PEM stays thicker) for crosslinked PEM in high and low pH values. In addition the morphological changes to a slightly smoother PEM (compare Fig. 5 c and d). The inner structures of PEM
films suffer not only a contraction, but a structural
change upon crosslinking as well. Estimating a simple contraction of the PEM, with a constant pore diameter wi the PEM, one would expect an increased diffusion through the PEM. Interestingly the contrary is the case, the diffusion decreased by factor 3.8±0.25 for simple small dye molecules like FITC as shown in Fig. 6. This decrease proves that small pores and voids are closed upon crosslinking, which might hint a
contraction of our PEM film, whereby the voids and pores are closed upon contraction. This would however only be possible if the voids would not lead in a straight way through the PEM which has an approximate thickness of 1 µm. The pores are usually in the nano-range and were until now only observed in the special case of strongly swollen PEM of low pH or extremely high salt concentration, where the PEM was already in state of being dissolved.[31,38] Alternatively a multi-dimensional contraction of the pores is also possible, however unlikely, since the core was dissolved after the crosslinking, so the contraction of the sides was limited, whereby a complete internal re-arrangement cannot be ruled out completely. Preliminary tests of the core being dissolved before crosslinking however showed no shrinking, which is probably due to the PEM being crosslinked and hardening faster than the PEM diffusion on large length scales. The fact that PEM hydrophobic moieties are usually aligned along the surface plane and are not isotropically oriented supports our theory that the pores are not leading in a straight line through the PEM but in an indirect way, resulting in an easy to shrink or close path[37]. The remaining question, which could not be solved, is weather just a large amount of pores were closed while direct ones staid open, or all channels simply decreased in diameter. Our pore closing theory is supported by SEM images, showing a smoother surface after crosslinking (see Fig. 5). 3.3 Drug delivery properties of the produced capsules Drug delivery experiments using these capsules showed that the crosslinked capsules are releasing a model drug (DOX) much slower compared to non crosslinked capsules as can be seen in Fig. 7. It is worth noting, that an asymptotic fit failed for this kind of sample and therefore an allomeric fit was used. Using an allometric fit (
, with x
being the time, Y being the released DOX in % and a and b being constants) to
determine the steepness of the release rate, one gets for non-crosslinked capsules a pre-exponent of 60 for a, while it is only 40 for crosslinked capsules. This difference in 50% in the pre-exponential factor is due to the increased diffusion time. Interestingly the exponential constant for the crosslinked samples is much higher than for the non-crosslinked samples (0.22 versus 0.08). This observation is owed by the fact that the non-crosslinked capsules released their DOX already before they could be measured in the UV spectrometer. Therefore these samples have a higher offset and a low increase since the plateau is already reached in the first half hour. The crosslinked samples on the contrary only lost 50% of their cargo and show therefore a stronger increase after the first 30 minutes. Comparing the increase in release after the first 30 minutes, we observe a difference in diffusion by factor 2.5 between crosslinked and non-crosslinked capsules, which are closer to the FITC diffusion values measured by FRAP in Fig 6. The fact, that the release rate does not reach 100% in our experiments is due to the fact that in the first washing steps a considerable amount (20-30%) of DOX is already released and removed. Although the drug release rate is in our case much faster and less easy to control as in case of pH triggered release[39] it provides a novel path to deliver drugs. We would like to point out, that our approach is especially interesting for cases, where the drug delivery is necessary, but no clear pH gradient is present. In such cases a diffusion dependent release might be better than a delivery system requiring a alkaline pH[39], which is present in parts of the human or cellular stomach systems. To investigate if our crosslinked capsules show a pH dependent change in diffusion coefficient (e.g. if smaller versions could be eaten in form of a therapy) we incubated crosslinked and non-crosslinked capsules into a FITC containing media at pH 3, 7, and
11. As shown in Fig. 9 the crosslinked capsules are pH responsive, whereby low pH causes an increased diffusion through the capsule wall, probably due to acid based disassembly of the PEs. In case of pH 7 the diffusion coefficient is lowest, while for pH 11 a slightly increased diffusion coefficient is observed. For non-crosslinked capsules the diffusion coefficient is high for all pH ranges. This finding is surprising and allows for utilization of our drug carriers in case of endosome or lysosome based drug release systems in the future.[40] To investigate the biodegradability of our novel photocrosslinkable drug delivery systems, we incubated crosslinked and non-crosslinked capsules in pancreatin solution and investigated the structural change of the capsules after one night. As can be seen in Fig. 10, both the crosslinked and non-crosslinked capsules were significantly degraded. The crosslinking reduced the degradation degree to some extent, but the biodegradability was preserved. The fact that a human body the temperature is 37 and not 20 °C, will allow a significantly higher degree of biodegradation than in this study. The capsules were neither in crosslinked nor in pristine form poisonous to HELA cells (Fig. 11 a). Upon embedding DOX into the capsules, the viability of the HELA cells could be significantly decreased depending on the amount of embedded DOX (Fig. 11 b). This proves the usability of the proposed drug delivery system.
4. Conclusion We successfully crosslinked PEM films comprising out of ALG and CHI. The crosslinking mechanism exhibits 2 components, which we suspect to stem from reactions between the vinyl groups and side reactions, from crosslinking with the backbone of the counter PE, since PE of the same kind are shielded from each other due
to charge repulsion. We also show for the first time, that it is possible to produce photo-crosslinked PEM capsules. These crosslinked PEM capsules show a decreased diffusion of dyes through their membrane compared to non crosslinked capsules of the same diameter and same composition. Drug delivery experiments showed a clear decrease in
the diffusion rate and release of DOX compared to non-crosslinked
counterparts. This allows a controlled release over longer time periods for example overnight. In case of placing recognition sites onto the capsules to detect tumor sites, the decreased release rate allows the capsules to stay a longer time in the body until they reach the tumor site due to the blood flow. The proposed system can easily be used to produce smaller capsules by simply using smaller templates. In addition the used PEM system is biodegradable yielding no poisonous substances.
Acknowledgements This work was financially supported by Natural Science Foundation of China (Grant No. 81170960),
Startup
Grant
of HIT,
China
Postdoctoral
Science
Foundation
(2013M531019) and Foundation of the Department of Science and Technology of Heilongjiang Province (Grant No. GC12C303-2).
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Figure captions:
Scheme 1. Structure of vinylbenzene (VB) chemically linked to Alginate
Fig. 1. UV absorption spectra of ALG-VB/CHI PEM films. Films without VB do not show the absorption peak at 252 nm. Fig. 2. Comparison of a) Extinction and b) inverse absorption of the VB absorption peak at 252 nm. The PEM grows single exponentially which is typical for this type of PEM films. Layers of the X axis in a) and b) correspond to mono layers of single PE. Lines in a) and b) are linear and exponential fits. Fig. 3. Zeta potential measurement of 1 µm capsules for several deposition cycles. Fig. 4. a) Absorption spectra of the 252 nm absorption peak of the vinyl group in ALG-VB upon UV irradiation. After 45 minutes of irradiation this peak is nearly totally gone; b) Intensity of the 252 nm absorption peak in extinction with a single exponential decay fit, whereby the inset shows the relative absorption with single and double exponential decay fits. Fig. 5. The SEM images of the capsule with (A, C, and E) and without UV irradiation (B, D, and F) in pH 3 (A and B), pH 7 (C and D), and pH 11 (E and F). Fig. 6. Comparison of the fluorescence recovery after photo-bleaching of FITC for a)-d) not crosslinked and e)-h) photo-crosslinked PEM capsules. The non crosslinked capsules recover the fluorescence faster as a comparison of the intensity versus time plots for non-crosslinked i) and crosslinked j) PEM capsules shows. Fig. 7. Drug (DOX) release properties of crosslinked and not crosslinked PEM capsules. a) Normalized release rate of PEM capsules, b) release rate versus time. The used allometric fit in a) and the exponential decay fit in b) show a clear discrepancies between crosslinked and non crosslinked capsules. The release in percent can be seen in Fig. 8. Fig. 8. Drug (DOX) release properties of crosslinked and not crosslinked PEM capsules.
The used allometric fit shows clear discrepancies between crosslinked and non crosslinked capsules. Fig. 9. The CLSM images of the cross linked capsules (A, B, and C) and non-cross linked capsule (D, E, and F) under pH 3 (A and D), pH 7 (B and E), and pH 11 (C and F). Scale bar 20 µm. Diffusion constant of capsule wall different for crosslinked and non-crosslinked ones as well as for different pH. Fig. 10. The SEM images of the capsule without treatment (A), under enzyme treatment (B), under UV irradiation (UV irradiation), and under the enzyme treatment and UV irradiation (D). Images taken after incubating the capsules for 1 night in pancreatin solution at room temperature. Fig. 11. Cells viability essay (MTT) of cells being incubated with (A) not crosslinked and crosslinked capsules. (B) Crosslinked capsules loading different concentration of DOX. As can be seen the drug can be successfully be delivered to HELA cells. Figures and Schemes H
H
H OH
HO COONa
OH
H H
OH
O H OH
O
H H OH
O
C O
O
Scheme 1.
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Fig. 11.