- Email: [email protected]
Smart Carriers for Controlled Drug Delivery: Thermosensitive Polymers Embedded in Ordered Mesoporous Carbon
Accepted Manuscript Smart carriers for controlled drug delivery: thermosensitive polymers embedded in ordered mesoporous carbon Meisam V. Kiamahalleh, Amir Mellati, S. Amirhossein Madani, Phillip Pendleton, Hu Zhang, S. Hadi Madani PII:
S0022-3549(17)30082-5
DOI:
10.1016/j.xphs.2017.02.010
Reference:
XPHS 652
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 20 December 2016 Revised Date:
23 January 2017
Accepted Date: 6 February 2017
Please cite this article as: Kiamahalleh MV, Mellati A, Madani SA, Pendleton P, Zhang H, Madani SH, Smart carriers for controlled drug delivery: thermosensitive polymers embedded in ordered mesoporous carbon, Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.xphs.2017.02.010. 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.
ACCEPTED MANUSCRIPT
Smart carriers for controlled drug delivery:
mesoporous carbon
RI PT
thermosensitive polymers embedded in ordered
a
M AN U
Madanid,e,*
SC
Meisam V. Kiamahalleha, Amir Mellatia,b, S. Amirhossein Madanic, Phillip Pendletona, Hu Zhanga, S. Hadi
School of Chemical Engineering, University of Adelaide, Adelaide, SA 5005, Australia b
Advanced Pharmaceutical Technologies Department, Tofigh Daru Research and Engineering Co., Tehran, 1397116395, Iran
TE D
Department of Mechanical Engineering, Iran University of Science and Technology, Tehran, 16765-163, Iran
Australian School of Petroleum, The University of Adelaide, Adelaide, SA 5005, Australia e
EP
d
Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095,
AC C
c
Australia. *
Corresponding author:
Dr. Hadi Madani, E-mail: [email protected], TEL: +61-8-8313-8044
ACCEPTED MANUSCRIPT KEYWORDS Thermosensitive polymer, PNIPAAm, Ordered mesoporous carbon, CMK3, Drug delivery, DOX
RI PT
Abstract An efficient drug delivery system was introduced. The carrier was synthesized by combination of an ordered mesoporous carbon (CMK3) and a thermosensitive polymer, Poly(N-isopropylacrylamide), known as PNIPAAm. The polymers with two different chain
SC
lengths (PNIPAAMm-100n and PNIPAAm 400n) were synthesized and each of the polymers was embedded in CMK3 to form composite materials. Nitrogen adsorption isotherm and
M AN U
scanning electron microscopy of the samples showed a uniform embedding of PNIPAAMm100n but a non-uniform embedding of PNIPAAMm-400n. The latter observation is attributed to large intra-molecular interactions of PNIPAAMm-400n and their aggregation on the external surface of the porous structure. Doxorubicin (DOX) was used as the model drug and was loaded onto the samples. The ultimate loading capacities for the polymer-embedded samples were reduced. However, the loading rates and the release capacities were
TE D
significantly improved. Thermosensitivity of the polymer was introduced as the governing drug release mechanism; regardless of the polymer chain length, drug release at 37 °C was significantly higher than 4 °C. Cytotoxicity results confirmed materials’ biocompatibility for future biological tests. It is clearly shown that the properly-synthesized composite of ordered
EP
mesoporous carbon and thermosensitive polymer can be used as an efficient carrier for drug loading and release experiments. The loading and release profiles can be controlled by
AC C
tailoring the polymer chain length. 1- Introduction
Drug delivery to the affected sites inside the body or into the living cells has been a fundamental target in pharmaceutical science and a very intense research field over the past four decades. The main aim of controlled drug delivery is to introduce new biomaterials to attain the ability of delivering drugs to pre-designated areas of the body and in pre-defined release rates to overcome the traditional drugs drawbacks
1-7
. Particularly, delivery of
hydrophobic drugs, due to their poor solubility, has been frequently studied in the literature over the past decade
8-10
. Various carriers have been used in the literature for drug delivery
ACCEPTED MANUSCRIPT studies. These carriers can be classified into two general categories: organic and inorganic carriers. Due to their availability, pore size flexibility, pore shape control, monitoring capability and tuneable surface functionalities, inorganic carriers have been more frequently applied for delivery studies mesoporous carbon
15
11-13
. Examples include, but not limited to mesoporous silica
, and composites
16
14
,
. Due to their strong adsorption capacity, chemical
RI PT
inertness, desirable pore size for drug adsorption, large specific surface area and pore volume, and consequently their potential for high drug loading, ordered mesoporous carbons (OMCs) are considered adorable carrier materials for drug delivery 17-19.
Different strategies have been developed to load drug molecules onto porous carbonaceous 18-22
. Chemical adsorption has
SC
materials either physically or chemically (via covalent bonds)
disadvantage: (1) covalent bonds between the drug molecules and mesoporous carbons are
M AN U
insufficient, as even a minor change in the molecular structure of a drug may drastically change its unique properties; (2) covalent bonds provide stronger adsorption and more stable drug delivery carrier, but at the same time, they usually exhibit limited drug release capacities. Therefore, physical adsorption is more favorable for drug adsorption/release studies. As an example of ordered mesoporous carbons, CMK3 is chosen here as the carrier of interest. CMK3 surface contains carbon aromatic rings, allowing interactions (including π–
TE D
π stacking) with hydrophobic drugs. However, the highly hydrophobic surface of CMK3 hinders its dispersion in physiological aqueous phases. Introducing oxygen group to the surface is an approach to offer hydrophilic characteristics, but the consequence is losing some density of π-electron clouds. Incorporation of hydrophilic polymers on the side walls of the
EP
CMK3 could be an alternative approach. Though, enhancing the drug-loading efficiency depending on the nature of the polymers remains a challenge in drug-carrier design.
AC C
To increase drug load capacity, a variety of the polymers is available to be introduced to the OMC structure. Due to their unique properties, stimuli-responsive polymers are attracted enormous attentions in the literature for different biological applications delivery
25,26
23,24
, including drug
. These smart materials can respond to external stimuli, such as temperature, pH,
ionic strength, light, magnetism, etc. in a controllable and predictable manner
27
. The
response is a change in some chemical or physical properties such as dissolution, precipitation, degradation, swelling, deswelling, shape, etc. Thermosensitive polymers, one the most commonly studied stimuli-responsive polymers, can undergo a reversible/irreversible phase transition in an aqueous environment at a certain
ACCEPTED MANUSCRIPT temperature. When the polymer aqueous solution is sufficiently concentrated, a sol-gel transition occurs while in case of cross-linked hydrogels, a swelling/shrinkage phenomenon happens. This thermo-responsive transition behavior makes this category of polymers a promising biomaterial in drug delivery 25. PNIPAAm is a well-known thermosensitive polymer.
The transition temperature of
RI PT
PNIPAAm, namely lower critical solution temperature (LCST), is around 31 ˚C which is close to the body temperature (37˚C). This unique characteristic has made PNIPAAm as one of the most interesting thermo-responsive polymers in biomedical application including drug delivery
28,29
. Drug-loaded carriers made from PNIPAAm or its copolymers, blends or
SC
hybrids with other materials collapse when administered into the body at physiological temperature and the drug is released. The release profile can be tailored by controlling the
M AN U
material properties through various chemical and physical modifications 30. Embedding porous substances with polymers is challenging. In situ polymerization technique have been widely used to embed 3,4-ethylenedioxythiophene (PEDOT)
31
and PNIPAAm
18
with mesoporous carbon. However, this brings two major shortcomings for the pore structure; (1) the mesoporous structure might be turned into a non-porous structure as the pores of
TE D
OMC might be filled with polymers, (2) there is no control on the polymer chain length (molecular weight), hence no control in pore size, the amount of drug uptake and release. In drug delivery systems, the porous structures are preferred to nonporous structures where the pores function as channels for drug release as well as a space for drug storage. In addition,
EP
the internal embedded polymer plays a key role as a storage gate as well as a release switch in response to the environmental stimuli. Zhu et al.
18
developed a new strategy for the
incorporation of the PNIPAAm inside the porous channels of CMK3 with claiming of not
AC C
altering their porous structures. However, controlling the pore size and volume remains a big challenge.
In this presentation, we aim to design the CMK3 sample conjugated with PNIPAAm that forms polymeric micelles on the side walls of the CMK3 and provides three-dimensional space to encapsulate a large amount of drugs in the carrier. We use PNIPAAm samples prepared through a well-controlled polymerization method which results in a narrowdispersed polymeric molecular weights with 100 (PNIPAAm-100n ) and 400 (PNIPAAm400n ) units in their chains. Doxorubicin (DOX), a common anticancer agent, was used as a sample drug due to its high density of π-electron clouds on its aromatic rings and hydrogen
ACCEPTED MANUSCRIPT bonding functionalities
32-36
that, respectively, favor its binding with CMK3 and PNIPAAm
in the conjugated structure. To overcome the pore filling issue and to better control the drug delivery system, we report a new idea on the synthesis of composite materials, combining CMK3 separately with PNIPAAm-100n and PNIPAAm-400n. Characterization of the composites shows a great control on the pore size. Taking advantages of PNIPAAm’s
RI PT
thermo-sensitivity, we eventually show promising results in controlling the uptake and release dosage of the drug. Biocompatibility of the material is examined via cytotoxicity test. Whilst a specific porous material and a specific thermosensitive polymer are considered, the methodologies and concepts used here are general and can both extend and complement the
SC
current literature. 2- Materials
M AN U
CMK3 carbon is a commercially available ordered mesoporous polymer-based carbon synthesized via pore filling the ordered mesoporous silica molecular sieve SBA-15 as a template
37
. The CMK3 sample used in this work was purchased from ACS Materials (US)
and used as received. For convenience, the CMK3 sample is denoted as CMK in this work. N-Isopropylacrylamide
(NIPAAm,
97%),
copper
chloride
(CuCl),
Tris[2-
TE D
(dimethylamino)ethyl]amine (Me6Tren, 97%) and ethyl 2-chloropropionate (ECP) were purchased from Sigma-Aldrich and NIPAAm was purified by recrystallization in n-hexane. Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, USA). 3-(4,5-
EP
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was ordered from Molecular Probes (Oregon, USA). All other chemicals were of analytical grades and used directly
AC C
without further purification.
Doxorubicin hydrochloride (C27H29NO11 ·HCl) was purchased from Sigma Aldrich and also used directly without further purification. 3- Methods
ATRP of PNIPAAm: In previous studies
38-40
, we prepared a range of PNIPAAms with different controlled chain
lengths using a well-established controlled/living radical polymerisation method, called Atom Transfer Radical Polymerization (ATRP)
41,42
. In summary, in a typical polymerisation of
ACCEPTED MANUSCRIPT PNIPAAm-400n with a target degree of polymerization of 400 (DPn=400), NIPAAM (4.0 g, 17.6 mmol), CuCl (8.7 mg, 0.088 mmol) and 2-propanol (2 mL) were introduced into a dried Schlenk flask and fitted with a septum. Three freeze–pump–thaw cycles were performed. Me6Tren (24 µl, 0.088 mmol) was added via a nitrogen-purged syringe and the solution was stirred for 40 min to allow formation of the CuCl/Me6TREN complex. 11 µl (0.088 mmol) of
RI PT
ECP was added using an air-free syringe to start the polymerisation at 25°C. The reaction was exposed to air after 8 h. The solution was diluted in tetrahydrofuran and passed through
filtered and dried. Polymer embedding on carbon structure
SC
an alumina column to remove the catalyst. The polymer was precipitated into diethyl ether,
Three CMK dispersions were initially prepared by dispersing 10 mg CMK in 10 mL MilliQ
M AN U
water containing 10 wt% ethanol with the aid of room temperature ultrasonication for about 15 min. One dispersion was kept as plain CMK sample and the other two underwent the treatment process with PNIPAAms. The same amount of PNIPAAm-100n and PNIPAAm400n (10 mg) was added in each of the CMK dispersions. The mixtures of CMK and PNIPAAms were kept stirring overnight for 24 hrs at the room temperature for better
TE D
dispersibility and uniform coating of polymer on CMK. The final composite materials containing PNIPAAm-100n and PNIPAAm-400n were then denoted as CMK-P1 and CMKP4, respectively.
EP
Drug adsorption/release experiments
DOX was considered as a sample anticancer drug in this study, due to its semihydrophobic/hydrophilic characteristics. The drug loading of DOX into the plain CMK,
AC C
CMK-P1and CMK-P4 composite material was conducted as follows: For each observation, 3 mL of DOX solution (50 µg/mL) was added into each dispersion as-prepared in the previous section, and stirred at room temperature for 24 hrs. Sampling for every two hours started immediately after adding DOX into the dispersion. Every time, 1 mL of the dispersion was taken, centrifuged and the light absorbance of supernatant containing DOX was monitored at a wavelength of 490 nm [187, 433] by a Shimadzu 1601 UV-Vis Spectrophotometer. After each absorbance reading the sample was returned to its own stirring dispersion. The amount of releasing DOX in PBS was calculated from the calibration curve shown in the supporting information (Supporting Figure 1).
ACCEPTED MANUSCRIPT To monitor the release of DOX from the CMK based materials, the DOX-loaded samples were rinsed a few times with MilliQ water to remove unbound drug and drug attached to the outer surface of the materials. Each DOX-loaded sample was then divided into two roughly equal parts for tracking the DOX release at 4 ˚C and 37 ˚C in order to investigate the temperature dependency of the drug release profile. Each part was placed into a container
RI PT
with 5 mL aqueous PBS solution at pH 7.4 to mimic the release profile in physiological neutral environments. The dispersions in the containers were constantly stirred whilst being maintained at 4 ˚C in the cold room and 37 ˚C in an incubator. At predetermined time intervals, 1 mL of the solution from the containers was withdrawn and centrifuged to
SC
determine the DOX release using UV-Vis spectroscopy. After each UV-Vis measurement the precipitated material was returned to its own container and the supernatant was discarded (with 1 mL of fresh PBS solution replacing it). The cumulative percentage release of the
M AN U
DOX from the CMK based materials at different temperatures was then determined. Nitrogen adsorption
Nitrogen gas adsorption experiments were carried out at 77 K using a Belsorp-max gas adsorption apparatus. Samples were degassed prior to the adsorption experiments at ambient
TE D
temperature and a background vacuum of 10-5 kPa for 10 hours. Ultra high purity (>99.999%) helium and nitrogen from Coregas Australia were used for dead-space measurements and adsorption experiments respectively.
EP
Scanning Electron Microscopy (SEM)
The surface morphology of the resulting CMK based materials was characterized by high resolution field emission scanning electron microscope (FEI-SEM Quanta 450). Samples
AC C
were all imaged at an accelerating voltage as low as 2 kV in order to avoid any perturbation or damage to the polymer structure. Cytotoxicity
HEK 293T was cultured in DMEM supplemented with 10% FBS, 100 U mL-1 of penicillin, 100 mg mL-1 of streptomycin and 2 mM L-1 glutamine. The cells were incubated at 37°C in a humidified atmosphere in the presence of 5% CO2. Cells were trypsinized, reseeded in 96well plates (200 µL per well) at a density of 5.0×104 cells/mL and allowed to adhere overnight. The growth medium was replaced with fresh medium (200 µL) containing CMK-
ACCEPTED MANUSCRIPT P1 or CMK-P4 at concentrations of 0.3125, 0.625, 1.25 and 2.5 mg/mL. Cells were then incubated for 48 h before measuring the cytotoxicity. Cell viabilities were examined using the MTT assay. 10 µL of MTT (5 mg/ml in PBS) were added to each well, including both samples and controls, and then incubated for 4 h at 37˚C. All the liquid was removed from wells and 150 µL dimethyl sulfoxide (DMSO) was added to
RI PT
each well to ensure complete solubilization of formazan crystals. After 1 h further incubation, the absorbance was read using an ELx808 microplate reader (BioTek, USA) at 595 nm. 4- Results and discussions
SC
Figure 1 shows a schematic representation of CMK pore structure (a), polymer-embedded samples, CMK-P1 (b) and CMK-P4 (c), and, drug adsorption on CMK sample and polymer-
M AN U
embedded samples (d-f). We appreciate that the actual CMK pore network is a three dimensional structure including of a variety of interconnected and/or disconnected complex pore geometries with heterogeneous surface and random defects. However, we believe the simple schematic in Figure 1 can be used for a better understanding of the polymer incorporation and drug adsorption mechanisms. Further discussion on this figure is provided
Nitrogen gas adsorption
TE D
in the next sections.
Figure 2 shows nitrogen adsorption isotherms on plain CMK, CMK-P1, and CMK-P4. Isotherms exhibit a relatively large contribution of micropores at low pressures, characterized
EP
with type I isotherm based on IUPAC classification, followed by a relatively large contribution of mesopores at high pressures, characterized by type IV adsorption isotherm
AC C
based on IUPAC classification 43. More characteristics data for these samples are reported in Table 1.
At medium and high pressures, each isotherm exhibits a relatively broad and wide Type H4 hysteresis loop based on IUPAC classification 43, usually representing capillary condensation which is an indication of presence of mesoporosity. Isotherms in Figure 2 and properties in Table 1 clearly show reduction in pore volume and surface area with polymer incorporation for both CMK-P4 and CMK-P1. This reduction is a proof that polymer molecules are not just covered the external surface of the porous media, but they are sufficiently small to accommodate inside the pores of CMK. This partial pore-
ACCEPTED MANUSCRIPT filling by polymers is schematically shown in Figure 1 (b,c). Based on the data in Table 1 mesopore volume fractions for the three samples are the same which means micropore and mesopore volumes are being reduced proportionally and both micropore and mesopores are being filled and/or blocked by polymer molecules. Although the same mass and mass ratio of CMK/polymer is used for both polymer-embedded samples, results in Figure 2 and Table 1
RI PT
show larger reduction in the pore volume and the surface area for CMK-P1 compared to CMK-P4. This large reduction means that more pore volume of CMK-P1 is occupied by the polymer compared to CMK-P4. Since the same mass of polymer was used, it can be concluded that either density of PNIPAAm-100n is lower than PNIPAAm-400n resulting in
SC
more pore volume filling, or PNIPAAm-400n is embedded non-uniformly on the CMK; it is more embedded on the external surface compared to PNIPAAm-100n. This issue is clearly
M AN U
addressed in morphology section.
Calculated pore size distributions (PSD) for plain and polymer-embedded CMK samples are reported in Figure 3. PSD for plain CMK shows a main peak centered at 5.3 (mesopore range). A small and narrow peak is also shown at ≈ 1nm (micropore range). CMK is synthesized by using SBA-15 as a template. The main peak in the mesopore range originates from the structure of SBA-15. The small and narrow peak in micropore range originates from
TE D
the inherent porosity created in carbon material as a result of activation. As it was expected from isotherms, incorporation of polymers on CMK surface results in reduction in intensity of peaks in the PSD. Again, this reduction is more pronounced for
EP
CMK-P1. Figure 3 illustrates that incorporation of polymers on the surface substantially reduces pore volumes, represented by the total area under the peaks. This reduction is consistent with the total pore volumes calculated and shown in Table 1. The main PSD peaks
AC C
are shifted to slightly smaller pore sizes for polymer-embedded samples and their intensities are also smaller compared to the original peaks in CMK PSD. This effect is related to occupying the porosity by the polymer molecules. Polymer is embedded across whole the pore geometry. As a consequence of uniform embedding, firstly, pore size decreases and the peaks are shifted to slightly smaller pore sizes; secondly, pore volume decreases which results in smaller intensity of peaks (shrinkage of peaks). This explanation is schematically shown in Figure 1 (b,c); for both cases (CMK-P1 and CMK-P4), incorporation of polymer with CMK results in smaller pore sizes and reduced pore volumes. This is why the calculated PSDs for polymer-embedded samples are identical (but smaller) compared to PSD for original sample.
ACCEPTED MANUSCRIPT Morphology The surface morphologies and microstructures of CMK carbon and polymer-embedded samples were examined by scanning electron microscopy (SEM). Figure 4 shows the SEM images of the (a) CMK mesoporous carbon, (b) CMK-P1, and (c) CMK-P4. It can be clearly seen that the plain CMK carbon sample is mainly made up of large rod-like particles with
RI PT
several micrometers in diameter, and big particles are composed of many sub-micrometersized aggregated rod-like particles with an average diameter of ~0.35 µm. As it is shown in Figure 4b, the original surface morphology was completely retained after modifying the CMK surface with PNIPAAm-100n. It made a uniform coating of polymer on the carbon 18,31
without any significant
SC
surface similarly to what have been observed in the literature
change in diameter of sub-micrometer-sized rods of CMK. We presume that almost all of
M AN U
PNIPAAm-100n without changing their elongated coil penetrated into the CMK mesoporous structure, filled in most of the pores, consistent with N2 adsorption results which show a very significant decrease in the pore volume. In contrast, incorporation of PNIPAAm-400n inside CMK formed such a special mesoporous composite, with evidently a big change in the morphology as shown in Figure 4c. On the surface of the composite, there seems to be a thick layer of outer coating compared to the plain CMK carbon, indicating that possibly there is a
TE D
strong intra-molecular interaction in PNIPAAm-400n, leading to deconforming the overall structure and making it a bigger particle so that it could not penetrate into CMK and also could not fill the pore as perfect as PNIPAAm-100n. This observation is consistent with N2 adsorption results which showed the bigger pore volume for CMK-P4 to that of CMK-P1. As
EP
a result, a considerable amount of PNIPAAm-400n with high intermolecular interaction aggregated on the surface of CMK made the rod-like particles thicker, though this
AC C
aggregation is kind of uniform (no clumps of PNIPAAm-400n is seen) along rod-like CMK particle only influencing in the rods’ thickness, as derived from Figure 4c. So the average diameter of the sub-micrometer-sized aggregated rod-like particles constituting the CMK-P4 composite increased to ~0.55 µm, which is clearly bigger than that of the small particles of CMK carbon and CMK-P1. This observation is schematically shown in Figure 1 (b,c). PNIPAAm-100n is uniformly embedded in the CMK porous structure resulting in significant pore filling and moderate external surface coverage. PNIPAAm-400n is also embedded inside the pore geometry, however, having a larger molecule and possibly stronger intra-molecular interactions, it is more embedded on the external surface. Drug loading
ACCEPTED MANUSCRIPT Drug loading capacities of CMK, CMK-P1 and CMK-P4 were examined for 24 hrs and the DOX adsorption profiles are shown in Figure 5. Drug loading profiles can be compared from two different points of view: drug loading rate and ultimate loading capacity. Ultimate loading capacity for each sample is a function of its pore volume. Regardless of the material, if the experiment is given enough time for adsorption, ultimately all the pores will be filled
RI PT
by the drug due to interaction between the drug molecules and a continuous liquid drug phase would form inside the pores. According to Figure 5, CMK had the highest ultimate drug loading capacity (approximately twice of CMK-P1 and 1.5 time of CMK-P4). This order is consistent with the sample’s pore volume (CMK > CMK-P4 > CMK-P1). The ultimate drug
SC
loading capacities of the three samples are schematically shown in Figure 1 (d-f).
Drug loading rate is a function of available driving forces for adsorption. The driving force
M AN U
for loading on CMK is probably π–π stacking interactions between the aromatic rings of CMK3 and DOX, followed by condensation of drug inside the pores (pore filling). Despite its large ultimate loading capacity, the plain CMK sample has the slowest adsorption rate as the polymer-embedded samples have two extra adsorption driving force compared to that of plain CMK:
TE D
(1) Hydrogen bond between −OH of PNIPAAm and −OH of DOX, (2) Hydrogen bond between −COOH of PNIPAAm and −NH2 of DOX, The two additional driving forces result in higher adsorption rate at short and intermediate
EP
loading for polymer-embedded samples. This fast drug loading continues until all the available polymer and carbon surfaces are covered by drug (≈5 hrs). Once the polymer and the CMK surfaces are covered by the drug molecules, the loading mechanism is just pore
AC C
filling; loading rate at long loading time depends on the available pore volume. Drug release
Figure 6 shows the ratio of drug released in different time steps. For repeatability analysis, the experiments are measured three times and the data in Figure 6 are shown as average and standard deviations. The figure clearly shows that the dominant mechanism for drug release is polymer thermosensitivity; the release from polymer-embedded samples is significantly higher than plain CMK sample and the release rate quickly increased (for more than 3 times) with the increase of the temperature from 4 °C to 37 °C which is attributed to the thermosensitivity of PNIPAAm. PNIPAAm coil-like molecules are hydrophilic at 4 °C. They
ACCEPTED MANUSCRIPT turn to hydrophobic globule-like molecules at 37 °C which leads to the shrinkage of the structure. Consequently, the structure squeezes and pushes the drug out. In absence of polymer, the ultimate release of DOX from plain CMK sample, regardless of the temperature, was very low (less than 3%) due to hydrophobic interactions and supramolecular π–π stacking between DOX and CMK.
RI PT
The drug released from the CMK-P1 sample is significantly larger than CMK-P4. This observation somehow contradicts drug loading profiles in Figure 5; since CMK-P4 shows a higher loading capacity, one would expect it to show a higher drug release.
SC
Drug release from polymer-embedded samples follows a two-step mechanism: drug desorption from the polymer surface and the molecular diffusion inside the pore. The former step is well studied in the literature. At elevated temperatures, above LCST, the polymer 48,49
M AN U
shows hydrophobic properties and hydrogen bonds between the drug and the polymer break . The latter step is somehow challenging. At elevated temperatures drug releases from the
polymer surface. At the same time, high temperature enhances drug diffusion within the porosity. However, this enhancement in diffusion coefficient is also valid for plain CMK. A probable explanation for drug release mechanism is desorption from the polymer surface as
TE D
a result of thermosensitivity followed by enhancement in diffusion induced by combined effect of high temperature and turbulence in the pore due to polymer shrinkage. This turbulence created convective mass transfer driving force and significantly enhances overall drug release. Based on this proposed mechanism, the release is mainly governed by
EP
thermosensitivity of the polymer; the more the volume of polymer, the more the release volume. Confirmed by nitrogen adsorption and SEM results for the CMK-P1 more pore
AC C
volume is occupied by the polymer. Consequently, effect of thermosensitivity and drug release behavior is more pronounced for CMK-P1. This is why the burst release, and consequently the overall release for CMK-P1 are reasonably higher than that of CMK-P4. Cytotoxicity
The cell viabilities from the MTT assays are shown in Figure 7 by incubating HEK 293T cells with CMK-P1 and CMK-P4 over a range of concentrations up to 2.5 mg/mL for 48 h. Results revealed that for CMK-P1, cell survival was approximately more than 80% over the test concentration range. However, the viability decreased slightly by increasing the concentration. Cell viability decreased from 80% at 0.3125 mg/mL to 60% at 2.5 mg/mL for
ACCEPTED MANUSCRIPT CMK-P4. These results suggest low in vitro cytotoxicity as it represents a much higher intravenous material dose than required for in vivo drug delivery 50 and comparable with the cytotoxicity of other nano- or micro carriers for drug delivery 51. Based on our previous work 39
, the lower cell viability in case of CMK-P4 is not due to the intrinsic toxicity of the
polymer. PNIPAAm-400n precipitates at 28 ˚C and the culture temperature is 37 ˚C which is
RI PT
9 ˚C higher than the polymer LCST. Polymer chain entanglements result in a barrier which covers the cells’ surface and it could get more dense and impermeable with a stronger entanglement and less pore sizes by continuing temperature rise above the LCST which subsequently hurdle nutrients and oxygen delivery to the cells.
SC
5- Conclusion
In summary, a properly-synthesized composite of OMC and thermosensitive polymer was
M AN U
shown to be a desirable carrier for drug delivery purposes. Incorporation of polymers in CMK was characterized by nitrogen adsorption isotherm and SEM, showing a uniform incorporation for PNIPAAm-100n, but a non-uniform one for PNIPAAm-400n. PNIPAAm400n had more residual on the external surface. Polymer-embedded samples showed less ultimate drug adsorption capacities, but significantly higher drug loading rates and drug
TE D
release capacities. The release capacity of the polymer-embedded samples was shown to be significantly improved with the temperature which is a result of polymer thermosensitivity. The polymer with the less chain length, PNIPAAm-100n, showed a smaller loading capacity compared to PNIPAAm-400n, but a reasonably higher release capacity. This behavior is
EP
attributed to the better pore filling of PNIPAAm-100n which results in a more effective release capacity. Cytotoxicity results confirm bio-compatibility of the synthesized materials
AC C
for possible future biological applications. Acknowledgements
Authors thank Dr. Moein Navvab Kashani and Dr. Hamideh Elekaei for sharing their facilities and for their assistance with the data analysis and interpretation. References 1. Gupta PK 1990. Drug targeting in cancer chemotherapy: a clinical perspective. Journal of pharmaceutical sciences 79(11):949-962. 2. Bae YH, Park K 2011. Targeted drug delivery to tumors: myths, reality and possibility. Journal of Controlled Release 153(3):198.
ACCEPTED MANUSCRIPT 3. Lu Y, Chen S 2004. Micro and nano-fabrication of biodegradable polymers for drug delivery. Advanced drug delivery reviews 56(11):1621-1633. 4. Qian KK, Bogner RH 2012. Application of mesoporous silicon dioxide and silicate in oral amorphous drug delivery systems. Journal of pharmaceutical sciences 101(2):444-463. 5. Nigmatullin R, Thomas P, Lukasiewicz B, Puthussery H, Roy I 2015. Polyhydroxyalkanoates, a family of natural polymers, and their applications in drug delivery. Journal of Chemical Technology and Biotechnology 90(7):1209-1221.
RI PT
6. Gupta P, Vermani K, Garg S 2002. Hydrogels: from controlled release to pHresponsive drug delivery. Drug discovery today 7(10):569-579. 7. Saylor DM, Kim CS, Patwardhan DV, Warren JA 2009. Modeling microstructure development and release kinetics in controlled drug release coatings. Journal of pharmaceutical sciences 98(1):169-186.
SC
8. Kim J-H, Kim Y-S, Kim S, Park JH, Kim K, Choi K, Chung H, Jeong SY, Park R-W, Kim I-S, Kwon IC 2006. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. Journal of Controlled Release 111(1–2):228-234.
M AN U
9. Wang Y, Zhao Q, Han N, Bai L, Li J, Liu J, Che E, Hu L, Zhang Q, Jiang T, Wang S 2015. Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine 11(2):313-327. 10. Dong K, Liu Z, Li Z, Ren J, Qu X 2013. Hydrophobic Anticancer Drug Delivery by a 980 nm Laser-Driven Photothermal Vehicle for Efficient Synergistic Therapy of Cancer Cells In Vivo. Advanced Materials 25(32):4452-4458. 11. Zhao M-X, Zeng E-Z, Zhu B-J 2015. The Biological Applications of Inorganic Nanoparticle Drug Carriers. ChemNanoMat 1(2):82-91.
TE D
12. Ma Pa, Xiao H, Li C, Dai Y, Cheng Z, Hou Z, Lin J 2015. Inorganic nanocarriers for platinum drug delivery. Materials Today 18(10):554-564.
EP
13. Son SJ, Bai X, Lee SB 2007. Inorganic hollow nanoparticles and nanotubes in nanomedicine: Part 2: Imaging, diagnostic, and therapeutic applications. Drug Discovery Today 12(15–16):657-663.
AC C
14. Hu Y, Zhi Z, Wang T, Jiang T, Wang S 2011. Incorporation of indomethacin nanoparticles into 3-D ordered macroporous silica for enhanced dissolution and reduced gastric irritancy. Eur J Pharm Biopharm 79(3):544-551. 15. Ouyang Y, Shi HM, Fu RW, Wu DC 2013. Highly Monodisperse Microporous Polymeric and Carbonaceous Nanospheres with Multifunctional Properties. Sci Rep-Uk 3. 16. Swarnalatha S, Selvi PK, Ganesh Kumar A, Sekaran G 2008. Nanoemulsion drug delivery by ketene based polyester synthesized using electron rich carbon/silica composite surface. Colloids and Surfaces B: Biointerfaces 65(2):292-299. 17. Zhu W, Zhao Q, Zheng X, Zhang Z, Jiang T, Li Y, Wang S 2014. Mesoporous carbon as a carrier for celecoxib: The improved inhibition effect on MDA-MB-231 cells migration and invasion. Asian Journal of Pharmaceutical Sciences 9(2):82-91. 18. Zhu SM, Chen CX, Chen ZX, Liu XY, Li Y, Shi Y, Zhang D 2011. Thermoresponsive polymer-functionalized mesoporous carbon for controlled drug release. Mater Chem Phys 126(1-2):357-363.
ACCEPTED MANUSCRIPT 19. Kim T-W, Chung P-W, Slowing II, Tsunoda M, Yeung ES, Lin VSY 2008. Structurally Ordered Mesoporous Carbon Nanoparticles as Transmembrane Delivery Vehicle in Human Cancer Cells. Nano Letters 8(11):3724-3727. 20. Wu W, Wieckowski S, Pastorin G, Benincasa M, Klumpp C, Briand J-P, Gennaro R, Prato M, Bianco A 2005. Targeted Delivery of Amphotericin B to Cells by Using Functionalized Carbon Nanotubes. Angewandte Chemie International Edition 44(39):63586362.
RI PT
21. Pastorin G, Wu W, Wieckowski S, Briand J-P, Kostarelos K, Prato M, Bianco A 2006. Double functionalisation of carbon nanotubes for multimodal drug delivery. Chem Commun (11):1182-1184. 22. Bhirde AA, Patel V, Gavard J, Zhang G, Sousa AA, Masedunskas A, Leapman RD, Weigert R, Gutkind JS, Rusling JF 2009. Targeted Killing of Cancer Cells in Vivo and in Vitro with EGF-Directed Carbon Nanotube-Based Drug Delivery. ACS Nano 3(2):307-316.
SC
23. Mano JF 2008. Stimuli-Responsive Polymeric Systems for Biomedical Applications. Advanced Engineering Materials 10(6):515-527.
M AN U
24. Alarcon CdlH, Pennadam S, Alexander C 2005. Stimuli responsive polymers for biomedical applications. Chemical Society Reviews 34(3):276-285. 25. Kost J, Langer R 2001. Responsive polymeric delivery systems. Advanced Drug Delivery Reviews 46(1–3):125-148. 26. Qiu Y, Park K 2001. Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews 53(3):321-339. 27. Liu F, Urban MW 2010. Recent advances and challenges in designing stimuliresponsive polymers. Progress in Polymer Science 35(1–2):3-23.
TE D
28. Kjøniksen AL, Calejo MT, Zhu K, Cardoso AMS, de Lima MCP, Jurado AS, Nyström B, Sande SA 2014. Sustained Release of Naltrexone from Poly (N‐ Isopropylacrylamide) Microgels. Journal of pharmaceutical sciences 103(1):227-234.
EP
29. Wang Y, Xu H, Wang J, Ge L, Zhu J 2014. Development of a Thermally Responsive Nanogel Based on Chitosan–Poly (N‐Isopropylacrylamide‐co‐Acrylamide) for Paclitaxel Delivery. Journal of pharmaceutical sciences 103(7):2012-2021.
AC C
30. Rzaev ZMO, Dinçer S, Pişkin E 2007. Functional copolymers of Nisopropylacrylamide for bioengineering applications. Progress in Polymer Science 32(5):534-595. 31. Wang J, Yu X, Li Y, Liu Q 2007. Poly(3,4-ethylenedioxythiophene)/Mesoporous Carbon Composite. The Journal of Physical Chemistry C 111(49):18073-18077. 32. Wu S, Zhao X, Li Y, Du Q, Sun J, Wang Y, Wang X, Xia Y, Wang Z, Xia L 2013. Adsorption properties of doxorubicin hydrochloride onto graphene oxide: Equilibrium, kinetic and thermodynamic studies. Materials 6(5):2026-2042. 33. Yang X, Zhang X, Liu Z, Ma Y, Huang Y, Chen Y 2008. High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide. The Journal of Physical Chemistry C 112(45):17554-17558. 34. Shen H, Zhang L, Liu M, Zhang Z 2012. Biomedical applications of graphene. Theranostics 2(3):283-294.
ACCEPTED MANUSCRIPT 35. Zhang L, Xia J, Zhao Q, Liu L, Zhang Z 2010. Functional Graphene Oxide as a Nanocarrier for Controlled Loading and Targeted Delivery of Mixed Anticancer Drugs. Small 6(4):537-544. 36. Song E, Han W, Li C, Cheng D, Li L, Liu L, Zhu G, Song Y, Tan W 2014. Hyaluronic Acid-Decorated Graphene Oxide Nanohybrids as Nanocarriers for Targeted and pH-Responsive Anticancer Drug Delivery. ACS Applied Materials & Interfaces 6(15):11882-11890.
RI PT
37. Zhao D, Feng J, Huo Q, Melosh N, Fredrickson GH, Chmelka BF, Stucky GD 1998. Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Science 279(5350):548-552.
SC
38. Mellati A, Kiamahalleh MV, Madani SH, Dai S, Bi J, Jin B, Zhang H 2016. Poly(Nisopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering. Journal of Biomedical Materials Research Part A 104(11):2764-2774.
M AN U
39. Mellati A, Valizadeh Kiamahalleh M, Dai S, Bi J, Jin B, Zhang H 2016. Influence of polymer molecular weight on the in vitro cytotoxicity of poly (N-isopropylacrylamide). Materials Science and Engineering: C 59:509-513. 40. Mellati A, Fan C-M, Tamayol A, Annabi N, Dai S, Bi J, Jin B, Xian C, Khademhosseini A, Zhang H 2016. Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering. Biotechnology and Bioengineering:n/a-n/a. 41. Matyjaszewski K, Xia J 2001. Atom Transfer Radical Polymerization. Chemical Reviews 101(9):2921-2990.
TE D
42. Patten TE, Matyjaszewski K 1998. Atom Transfer Radical Polymerization and the Synthesis of Polymeric Materials. Advanced Materials 10(12):901-915. 43. Sing KSW, Everett DH, Haul RAW, Moscou L, Pierotti RA, Rouquerol J, Siemieniewska T 1985. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity. Pure Appl Chem 57(4):603.
EP
44. Brunauer S, Emmett PH, Teller E 1938. Adsorption of Gases in Multimolecular Layers. Journal of the American Chemical Society 60(2):309-319.
AC C
45. Sing KSW, Rouquerol F, Llewellyn P, Rouquerol J. 2014. 9 - Assessment of Microporosity. In Maurin FRRSWSL, editor Adsorption by Powders and Porous Solids (Second Edition), ed., Oxford: Academic Press. p 303-320. 46. Sing KSW, Rouquerol F, Rouquerol J, Llewellyn P. 2014. 8 - Assessment of Mesoporosity. In Maurin FRRSWSL, editor Adsorption by Powders and Porous Solids (Second Edition), ed., Oxford: Academic Press. p 269-302. 47. Neimark AV, Lin Y, Ravikovitch PI, Thommes M 2009. Quenched solid density functional theory and pore size analysis of micro-mesoporous carbons. Carbon 47(7):16171628. 48. Zhu S, Chen C, Chen Z, Liu X, Li Y, Shi Y, Zhang D 2011. Thermo-responsive polymer-functionalized mesoporous carbon for controlled drug release. Materials Chemistry and Physics 126(1–2):357-363.
ACCEPTED MANUSCRIPT 49. Deshmukh S, Mooney DA, McDermott T, Kulkarni S, Don MacElroy JM 2009. Molecular modeling of thermo-responsive hydrogels: observation of lower critical solution temperature. Soft Matter 5(7):1514-1521. 50. Chan JM, Zhang L, Yuet KP, Liao G, Rhee J-W, Langer R, Farokhzad OC 2009. PLGA–lecithin–PEG core–shell nanoparticles for controlled drug delivery. Biomaterials 30(8):1627-1634.
AC C
EP
TE D
M AN U
SC
RI PT
51. Horcajada P, Chalati T, Serre C, Gillet B, Sebrie C, Baati T, Eubank JF, Heurtaux D, Clayette P, Kreuz C, Chang J-S, Hwang YK, Marsaud V, Bories P-N, Cynober L, Gil S, Ferey G, Couvreur P, Gref R 2010. Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging. Nat Mater 9(2):172-178.
ACCEPTED MANUSCRIPT
Table 1. Structural properties of plain CMK and polymer-embedded CMK samples: CMK-P4 and CMK-P1 Micropore
Total pore
Mesopore
surface area*
volume**
volume***
volume fraction
(cm2/g)
(cm3/g)
(cm3/g)
(%)
CMK
1055
0.34
1.41
0.76
CMK-P4
637
0.21
0.75
0.72
CMK-P1
489
0.15
SC
Sample ID
RI PT
BET equivalent
0.63
AC C
EP
TE D
M AN U
* Surface area is calculated based on BET method 44. **Micropore volume is calculated based on DR method 45. ***Total pore volume is calculated based on liquid volume adsorbed at p/po≈1 46.
0.76
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S0022-3549(17)30082-5
DOI:
10.1016/j.xphs.2017.02.010
Reference:
XPHS 652
To appear in:
Journal of Pharmaceutical Sciences
Received Date: 20 December 2016 Revised Date:
23 January 2017
Accepted Date: 6 February 2017
Please cite this article as: Kiamahalleh MV, Mellati A, Madani SA, Pendleton P, Zhang H, Madani SH, Smart carriers for controlled drug delivery: thermosensitive polymers embedded in ordered mesoporous carbon, Journal of Pharmaceutical Sciences (2017), doi: 10.1016/j.xphs.2017.02.010. 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.
ACCEPTED MANUSCRIPT
Smart carriers for controlled drug delivery:
mesoporous carbon
RI PT
thermosensitive polymers embedded in ordered
a
M AN U
Madanid,e,*
SC
Meisam V. Kiamahalleha, Amir Mellatia,b, S. Amirhossein Madanic, Phillip Pendletona, Hu Zhanga, S. Hadi
School of Chemical Engineering, University of Adelaide, Adelaide, SA 5005, Australia b
Advanced Pharmaceutical Technologies Department, Tofigh Daru Research and Engineering Co., Tehran, 1397116395, Iran
TE D
Department of Mechanical Engineering, Iran University of Science and Technology, Tehran, 16765-163, Iran
Australian School of Petroleum, The University of Adelaide, Adelaide, SA 5005, Australia e
EP
d
Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095,
AC C
c
Australia. *
Corresponding author:
Dr. Hadi Madani, E-mail: [email protected], TEL: +61-8-8313-8044
ACCEPTED MANUSCRIPT KEYWORDS Thermosensitive polymer, PNIPAAm, Ordered mesoporous carbon, CMK3, Drug delivery, DOX
RI PT
Abstract An efficient drug delivery system was introduced. The carrier was synthesized by combination of an ordered mesoporous carbon (CMK3) and a thermosensitive polymer, Poly(N-isopropylacrylamide), known as PNIPAAm. The polymers with two different chain
SC
lengths (PNIPAAMm-100n and PNIPAAm 400n) were synthesized and each of the polymers was embedded in CMK3 to form composite materials. Nitrogen adsorption isotherm and
M AN U
scanning electron microscopy of the samples showed a uniform embedding of PNIPAAMm100n but a non-uniform embedding of PNIPAAMm-400n. The latter observation is attributed to large intra-molecular interactions of PNIPAAMm-400n and their aggregation on the external surface of the porous structure. Doxorubicin (DOX) was used as the model drug and was loaded onto the samples. The ultimate loading capacities for the polymer-embedded samples were reduced. However, the loading rates and the release capacities were
TE D
significantly improved. Thermosensitivity of the polymer was introduced as the governing drug release mechanism; regardless of the polymer chain length, drug release at 37 °C was significantly higher than 4 °C. Cytotoxicity results confirmed materials’ biocompatibility for future biological tests. It is clearly shown that the properly-synthesized composite of ordered
EP
mesoporous carbon and thermosensitive polymer can be used as an efficient carrier for drug loading and release experiments. The loading and release profiles can be controlled by
AC C
tailoring the polymer chain length. 1- Introduction
Drug delivery to the affected sites inside the body or into the living cells has been a fundamental target in pharmaceutical science and a very intense research field over the past four decades. The main aim of controlled drug delivery is to introduce new biomaterials to attain the ability of delivering drugs to pre-designated areas of the body and in pre-defined release rates to overcome the traditional drugs drawbacks
1-7
. Particularly, delivery of
hydrophobic drugs, due to their poor solubility, has been frequently studied in the literature over the past decade
8-10
. Various carriers have been used in the literature for drug delivery
ACCEPTED MANUSCRIPT studies. These carriers can be classified into two general categories: organic and inorganic carriers. Due to their availability, pore size flexibility, pore shape control, monitoring capability and tuneable surface functionalities, inorganic carriers have been more frequently applied for delivery studies mesoporous carbon
15
11-13
. Examples include, but not limited to mesoporous silica
, and composites
16
14
,
. Due to their strong adsorption capacity, chemical
RI PT
inertness, desirable pore size for drug adsorption, large specific surface area and pore volume, and consequently their potential for high drug loading, ordered mesoporous carbons (OMCs) are considered adorable carrier materials for drug delivery 17-19.
Different strategies have been developed to load drug molecules onto porous carbonaceous 18-22
. Chemical adsorption has
SC
materials either physically or chemically (via covalent bonds)
disadvantage: (1) covalent bonds between the drug molecules and mesoporous carbons are
M AN U
insufficient, as even a minor change in the molecular structure of a drug may drastically change its unique properties; (2) covalent bonds provide stronger adsorption and more stable drug delivery carrier, but at the same time, they usually exhibit limited drug release capacities. Therefore, physical adsorption is more favorable for drug adsorption/release studies. As an example of ordered mesoporous carbons, CMK3 is chosen here as the carrier of interest. CMK3 surface contains carbon aromatic rings, allowing interactions (including π–
TE D
π stacking) with hydrophobic drugs. However, the highly hydrophobic surface of CMK3 hinders its dispersion in physiological aqueous phases. Introducing oxygen group to the surface is an approach to offer hydrophilic characteristics, but the consequence is losing some density of π-electron clouds. Incorporation of hydrophilic polymers on the side walls of the
EP
CMK3 could be an alternative approach. Though, enhancing the drug-loading efficiency depending on the nature of the polymers remains a challenge in drug-carrier design.
AC C
To increase drug load capacity, a variety of the polymers is available to be introduced to the OMC structure. Due to their unique properties, stimuli-responsive polymers are attracted enormous attentions in the literature for different biological applications delivery
25,26
23,24
, including drug
. These smart materials can respond to external stimuli, such as temperature, pH,
ionic strength, light, magnetism, etc. in a controllable and predictable manner
27
. The
response is a change in some chemical or physical properties such as dissolution, precipitation, degradation, swelling, deswelling, shape, etc. Thermosensitive polymers, one the most commonly studied stimuli-responsive polymers, can undergo a reversible/irreversible phase transition in an aqueous environment at a certain
ACCEPTED MANUSCRIPT temperature. When the polymer aqueous solution is sufficiently concentrated, a sol-gel transition occurs while in case of cross-linked hydrogels, a swelling/shrinkage phenomenon happens. This thermo-responsive transition behavior makes this category of polymers a promising biomaterial in drug delivery 25. PNIPAAm is a well-known thermosensitive polymer.
The transition temperature of
RI PT
PNIPAAm, namely lower critical solution temperature (LCST), is around 31 ˚C which is close to the body temperature (37˚C). This unique characteristic has made PNIPAAm as one of the most interesting thermo-responsive polymers in biomedical application including drug delivery
28,29
. Drug-loaded carriers made from PNIPAAm or its copolymers, blends or
SC
hybrids with other materials collapse when administered into the body at physiological temperature and the drug is released. The release profile can be tailored by controlling the
M AN U
material properties through various chemical and physical modifications 30. Embedding porous substances with polymers is challenging. In situ polymerization technique have been widely used to embed 3,4-ethylenedioxythiophene (PEDOT)
31
and PNIPAAm
18
with mesoporous carbon. However, this brings two major shortcomings for the pore structure; (1) the mesoporous structure might be turned into a non-porous structure as the pores of
TE D
OMC might be filled with polymers, (2) there is no control on the polymer chain length (molecular weight), hence no control in pore size, the amount of drug uptake and release. In drug delivery systems, the porous structures are preferred to nonporous structures where the pores function as channels for drug release as well as a space for drug storage. In addition,
EP
the internal embedded polymer plays a key role as a storage gate as well as a release switch in response to the environmental stimuli. Zhu et al.
18
developed a new strategy for the
incorporation of the PNIPAAm inside the porous channels of CMK3 with claiming of not
AC C
altering their porous structures. However, controlling the pore size and volume remains a big challenge.
In this presentation, we aim to design the CMK3 sample conjugated with PNIPAAm that forms polymeric micelles on the side walls of the CMK3 and provides three-dimensional space to encapsulate a large amount of drugs in the carrier. We use PNIPAAm samples prepared through a well-controlled polymerization method which results in a narrowdispersed polymeric molecular weights with 100 (PNIPAAm-100n ) and 400 (PNIPAAm400n ) units in their chains. Doxorubicin (DOX), a common anticancer agent, was used as a sample drug due to its high density of π-electron clouds on its aromatic rings and hydrogen
ACCEPTED MANUSCRIPT bonding functionalities
32-36
that, respectively, favor its binding with CMK3 and PNIPAAm
in the conjugated structure. To overcome the pore filling issue and to better control the drug delivery system, we report a new idea on the synthesis of composite materials, combining CMK3 separately with PNIPAAm-100n and PNIPAAm-400n. Characterization of the composites shows a great control on the pore size. Taking advantages of PNIPAAm’s
RI PT
thermo-sensitivity, we eventually show promising results in controlling the uptake and release dosage of the drug. Biocompatibility of the material is examined via cytotoxicity test. Whilst a specific porous material and a specific thermosensitive polymer are considered, the methodologies and concepts used here are general and can both extend and complement the
SC
current literature. 2- Materials
M AN U
CMK3 carbon is a commercially available ordered mesoporous polymer-based carbon synthesized via pore filling the ordered mesoporous silica molecular sieve SBA-15 as a template
37
. The CMK3 sample used in this work was purchased from ACS Materials (US)
and used as received. For convenience, the CMK3 sample is denoted as CMK in this work. N-Isopropylacrylamide
(NIPAAm,
97%),
copper
chloride
(CuCl),
Tris[2-
TE D
(dimethylamino)ethyl]amine (Me6Tren, 97%) and ethyl 2-chloropropionate (ECP) were purchased from Sigma-Aldrich and NIPAAm was purified by recrystallization in n-hexane. Dulbecco's Modified Eagle's Medium (DMEM), trypsin-EDTA, penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, USA). 3-(4,5-
EP
Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was ordered from Molecular Probes (Oregon, USA). All other chemicals were of analytical grades and used directly
AC C
without further purification.
Doxorubicin hydrochloride (C27H29NO11 ·HCl) was purchased from Sigma Aldrich and also used directly without further purification. 3- Methods
ATRP of PNIPAAm: In previous studies
38-40
, we prepared a range of PNIPAAms with different controlled chain
lengths using a well-established controlled/living radical polymerisation method, called Atom Transfer Radical Polymerization (ATRP)
41,42
. In summary, in a typical polymerisation of
ACCEPTED MANUSCRIPT PNIPAAm-400n with a target degree of polymerization of 400 (DPn=400), NIPAAM (4.0 g, 17.6 mmol), CuCl (8.7 mg, 0.088 mmol) and 2-propanol (2 mL) were introduced into a dried Schlenk flask and fitted with a septum. Three freeze–pump–thaw cycles were performed. Me6Tren (24 µl, 0.088 mmol) was added via a nitrogen-purged syringe and the solution was stirred for 40 min to allow formation of the CuCl/Me6TREN complex. 11 µl (0.088 mmol) of
RI PT
ECP was added using an air-free syringe to start the polymerisation at 25°C. The reaction was exposed to air after 8 h. The solution was diluted in tetrahydrofuran and passed through
filtered and dried. Polymer embedding on carbon structure
SC
an alumina column to remove the catalyst. The polymer was precipitated into diethyl ether,
Three CMK dispersions were initially prepared by dispersing 10 mg CMK in 10 mL MilliQ
M AN U
water containing 10 wt% ethanol with the aid of room temperature ultrasonication for about 15 min. One dispersion was kept as plain CMK sample and the other two underwent the treatment process with PNIPAAms. The same amount of PNIPAAm-100n and PNIPAAm400n (10 mg) was added in each of the CMK dispersions. The mixtures of CMK and PNIPAAms were kept stirring overnight for 24 hrs at the room temperature for better
TE D
dispersibility and uniform coating of polymer on CMK. The final composite materials containing PNIPAAm-100n and PNIPAAm-400n were then denoted as CMK-P1 and CMKP4, respectively.
EP
Drug adsorption/release experiments
DOX was considered as a sample anticancer drug in this study, due to its semihydrophobic/hydrophilic characteristics. The drug loading of DOX into the plain CMK,
AC C
CMK-P1and CMK-P4 composite material was conducted as follows: For each observation, 3 mL of DOX solution (50 µg/mL) was added into each dispersion as-prepared in the previous section, and stirred at room temperature for 24 hrs. Sampling for every two hours started immediately after adding DOX into the dispersion. Every time, 1 mL of the dispersion was taken, centrifuged and the light absorbance of supernatant containing DOX was monitored at a wavelength of 490 nm [187, 433] by a Shimadzu 1601 UV-Vis Spectrophotometer. After each absorbance reading the sample was returned to its own stirring dispersion. The amount of releasing DOX in PBS was calculated from the calibration curve shown in the supporting information (Supporting Figure 1).
ACCEPTED MANUSCRIPT To monitor the release of DOX from the CMK based materials, the DOX-loaded samples were rinsed a few times with MilliQ water to remove unbound drug and drug attached to the outer surface of the materials. Each DOX-loaded sample was then divided into two roughly equal parts for tracking the DOX release at 4 ˚C and 37 ˚C in order to investigate the temperature dependency of the drug release profile. Each part was placed into a container
RI PT
with 5 mL aqueous PBS solution at pH 7.4 to mimic the release profile in physiological neutral environments. The dispersions in the containers were constantly stirred whilst being maintained at 4 ˚C in the cold room and 37 ˚C in an incubator. At predetermined time intervals, 1 mL of the solution from the containers was withdrawn and centrifuged to
SC
determine the DOX release using UV-Vis spectroscopy. After each UV-Vis measurement the precipitated material was returned to its own container and the supernatant was discarded (with 1 mL of fresh PBS solution replacing it). The cumulative percentage release of the
M AN U
DOX from the CMK based materials at different temperatures was then determined. Nitrogen adsorption
Nitrogen gas adsorption experiments were carried out at 77 K using a Belsorp-max gas adsorption apparatus. Samples were degassed prior to the adsorption experiments at ambient
TE D
temperature and a background vacuum of 10-5 kPa for 10 hours. Ultra high purity (>99.999%) helium and nitrogen from Coregas Australia were used for dead-space measurements and adsorption experiments respectively.
EP
Scanning Electron Microscopy (SEM)
The surface morphology of the resulting CMK based materials was characterized by high resolution field emission scanning electron microscope (FEI-SEM Quanta 450). Samples
AC C
were all imaged at an accelerating voltage as low as 2 kV in order to avoid any perturbation or damage to the polymer structure. Cytotoxicity
HEK 293T was cultured in DMEM supplemented with 10% FBS, 100 U mL-1 of penicillin, 100 mg mL-1 of streptomycin and 2 mM L-1 glutamine. The cells were incubated at 37°C in a humidified atmosphere in the presence of 5% CO2. Cells were trypsinized, reseeded in 96well plates (200 µL per well) at a density of 5.0×104 cells/mL and allowed to adhere overnight. The growth medium was replaced with fresh medium (200 µL) containing CMK-
ACCEPTED MANUSCRIPT P1 or CMK-P4 at concentrations of 0.3125, 0.625, 1.25 and 2.5 mg/mL. Cells were then incubated for 48 h before measuring the cytotoxicity. Cell viabilities were examined using the MTT assay. 10 µL of MTT (5 mg/ml in PBS) were added to each well, including both samples and controls, and then incubated for 4 h at 37˚C. All the liquid was removed from wells and 150 µL dimethyl sulfoxide (DMSO) was added to
RI PT
each well to ensure complete solubilization of formazan crystals. After 1 h further incubation, the absorbance was read using an ELx808 microplate reader (BioTek, USA) at 595 nm. 4- Results and discussions
SC
Figure 1 shows a schematic representation of CMK pore structure (a), polymer-embedded samples, CMK-P1 (b) and CMK-P4 (c), and, drug adsorption on CMK sample and polymer-
M AN U
embedded samples (d-f). We appreciate that the actual CMK pore network is a three dimensional structure including of a variety of interconnected and/or disconnected complex pore geometries with heterogeneous surface and random defects. However, we believe the simple schematic in Figure 1 can be used for a better understanding of the polymer incorporation and drug adsorption mechanisms. Further discussion on this figure is provided
Nitrogen gas adsorption
TE D
in the next sections.
Figure 2 shows nitrogen adsorption isotherms on plain CMK, CMK-P1, and CMK-P4. Isotherms exhibit a relatively large contribution of micropores at low pressures, characterized
EP
with type I isotherm based on IUPAC classification, followed by a relatively large contribution of mesopores at high pressures, characterized by type IV adsorption isotherm
AC C
based on IUPAC classification 43. More characteristics data for these samples are reported in Table 1.
At medium and high pressures, each isotherm exhibits a relatively broad and wide Type H4 hysteresis loop based on IUPAC classification 43, usually representing capillary condensation which is an indication of presence of mesoporosity. Isotherms in Figure 2 and properties in Table 1 clearly show reduction in pore volume and surface area with polymer incorporation for both CMK-P4 and CMK-P1. This reduction is a proof that polymer molecules are not just covered the external surface of the porous media, but they are sufficiently small to accommodate inside the pores of CMK. This partial pore-
ACCEPTED MANUSCRIPT filling by polymers is schematically shown in Figure 1 (b,c). Based on the data in Table 1 mesopore volume fractions for the three samples are the same which means micropore and mesopore volumes are being reduced proportionally and both micropore and mesopores are being filled and/or blocked by polymer molecules. Although the same mass and mass ratio of CMK/polymer is used for both polymer-embedded samples, results in Figure 2 and Table 1
RI PT
show larger reduction in the pore volume and the surface area for CMK-P1 compared to CMK-P4. This large reduction means that more pore volume of CMK-P1 is occupied by the polymer compared to CMK-P4. Since the same mass of polymer was used, it can be concluded that either density of PNIPAAm-100n is lower than PNIPAAm-400n resulting in
SC
more pore volume filling, or PNIPAAm-400n is embedded non-uniformly on the CMK; it is more embedded on the external surface compared to PNIPAAm-100n. This issue is clearly
M AN U
addressed in morphology section.
Calculated pore size distributions (PSD) for plain and polymer-embedded CMK samples are reported in Figure 3. PSD for plain CMK shows a main peak centered at 5.3 (mesopore range). A small and narrow peak is also shown at ≈ 1nm (micropore range). CMK is synthesized by using SBA-15 as a template. The main peak in the mesopore range originates from the structure of SBA-15. The small and narrow peak in micropore range originates from
TE D
the inherent porosity created in carbon material as a result of activation. As it was expected from isotherms, incorporation of polymers on CMK surface results in reduction in intensity of peaks in the PSD. Again, this reduction is more pronounced for
EP
CMK-P1. Figure 3 illustrates that incorporation of polymers on the surface substantially reduces pore volumes, represented by the total area under the peaks. This reduction is consistent with the total pore volumes calculated and shown in Table 1. The main PSD peaks
AC C
are shifted to slightly smaller pore sizes for polymer-embedded samples and their intensities are also smaller compared to the original peaks in CMK PSD. This effect is related to occupying the porosity by the polymer molecules. Polymer is embedded across whole the pore geometry. As a consequence of uniform embedding, firstly, pore size decreases and the peaks are shifted to slightly smaller pore sizes; secondly, pore volume decreases which results in smaller intensity of peaks (shrinkage of peaks). This explanation is schematically shown in Figure 1 (b,c); for both cases (CMK-P1 and CMK-P4), incorporation of polymer with CMK results in smaller pore sizes and reduced pore volumes. This is why the calculated PSDs for polymer-embedded samples are identical (but smaller) compared to PSD for original sample.
ACCEPTED MANUSCRIPT Morphology The surface morphologies and microstructures of CMK carbon and polymer-embedded samples were examined by scanning electron microscopy (SEM). Figure 4 shows the SEM images of the (a) CMK mesoporous carbon, (b) CMK-P1, and (c) CMK-P4. It can be clearly seen that the plain CMK carbon sample is mainly made up of large rod-like particles with
RI PT
several micrometers in diameter, and big particles are composed of many sub-micrometersized aggregated rod-like particles with an average diameter of ~0.35 µm. As it is shown in Figure 4b, the original surface morphology was completely retained after modifying the CMK surface with PNIPAAm-100n. It made a uniform coating of polymer on the carbon 18,31
without any significant
SC
surface similarly to what have been observed in the literature
change in diameter of sub-micrometer-sized rods of CMK. We presume that almost all of
M AN U
PNIPAAm-100n without changing their elongated coil penetrated into the CMK mesoporous structure, filled in most of the pores, consistent with N2 adsorption results which show a very significant decrease in the pore volume. In contrast, incorporation of PNIPAAm-400n inside CMK formed such a special mesoporous composite, with evidently a big change in the morphology as shown in Figure 4c. On the surface of the composite, there seems to be a thick layer of outer coating compared to the plain CMK carbon, indicating that possibly there is a
TE D
strong intra-molecular interaction in PNIPAAm-400n, leading to deconforming the overall structure and making it a bigger particle so that it could not penetrate into CMK and also could not fill the pore as perfect as PNIPAAm-100n. This observation is consistent with N2 adsorption results which showed the bigger pore volume for CMK-P4 to that of CMK-P1. As
EP
a result, a considerable amount of PNIPAAm-400n with high intermolecular interaction aggregated on the surface of CMK made the rod-like particles thicker, though this
AC C
aggregation is kind of uniform (no clumps of PNIPAAm-400n is seen) along rod-like CMK particle only influencing in the rods’ thickness, as derived from Figure 4c. So the average diameter of the sub-micrometer-sized aggregated rod-like particles constituting the CMK-P4 composite increased to ~0.55 µm, which is clearly bigger than that of the small particles of CMK carbon and CMK-P1. This observation is schematically shown in Figure 1 (b,c). PNIPAAm-100n is uniformly embedded in the CMK porous structure resulting in significant pore filling and moderate external surface coverage. PNIPAAm-400n is also embedded inside the pore geometry, however, having a larger molecule and possibly stronger intra-molecular interactions, it is more embedded on the external surface. Drug loading
ACCEPTED MANUSCRIPT Drug loading capacities of CMK, CMK-P1 and CMK-P4 were examined for 24 hrs and the DOX adsorption profiles are shown in Figure 5. Drug loading profiles can be compared from two different points of view: drug loading rate and ultimate loading capacity. Ultimate loading capacity for each sample is a function of its pore volume. Regardless of the material, if the experiment is given enough time for adsorption, ultimately all the pores will be filled
RI PT
by the drug due to interaction between the drug molecules and a continuous liquid drug phase would form inside the pores. According to Figure 5, CMK had the highest ultimate drug loading capacity (approximately twice of CMK-P1 and 1.5 time of CMK-P4). This order is consistent with the sample’s pore volume (CMK > CMK-P4 > CMK-P1). The ultimate drug
SC
loading capacities of the three samples are schematically shown in Figure 1 (d-f).
Drug loading rate is a function of available driving forces for adsorption. The driving force
M AN U
for loading on CMK is probably π–π stacking interactions between the aromatic rings of CMK3 and DOX, followed by condensation of drug inside the pores (pore filling). Despite its large ultimate loading capacity, the plain CMK sample has the slowest adsorption rate as the polymer-embedded samples have two extra adsorption driving force compared to that of plain CMK:
TE D
(1) Hydrogen bond between −OH of PNIPAAm and −OH of DOX, (2) Hydrogen bond between −COOH of PNIPAAm and −NH2 of DOX, The two additional driving forces result in higher adsorption rate at short and intermediate
EP
loading for polymer-embedded samples. This fast drug loading continues until all the available polymer and carbon surfaces are covered by drug (≈5 hrs). Once the polymer and the CMK surfaces are covered by the drug molecules, the loading mechanism is just pore
AC C
filling; loading rate at long loading time depends on the available pore volume. Drug release
Figure 6 shows the ratio of drug released in different time steps. For repeatability analysis, the experiments are measured three times and the data in Figure 6 are shown as average and standard deviations. The figure clearly shows that the dominant mechanism for drug release is polymer thermosensitivity; the release from polymer-embedded samples is significantly higher than plain CMK sample and the release rate quickly increased (for more than 3 times) with the increase of the temperature from 4 °C to 37 °C which is attributed to the thermosensitivity of PNIPAAm. PNIPAAm coil-like molecules are hydrophilic at 4 °C. They
ACCEPTED MANUSCRIPT turn to hydrophobic globule-like molecules at 37 °C which leads to the shrinkage of the structure. Consequently, the structure squeezes and pushes the drug out. In absence of polymer, the ultimate release of DOX from plain CMK sample, regardless of the temperature, was very low (less than 3%) due to hydrophobic interactions and supramolecular π–π stacking between DOX and CMK.
RI PT
The drug released from the CMK-P1 sample is significantly larger than CMK-P4. This observation somehow contradicts drug loading profiles in Figure 5; since CMK-P4 shows a higher loading capacity, one would expect it to show a higher drug release.
SC
Drug release from polymer-embedded samples follows a two-step mechanism: drug desorption from the polymer surface and the molecular diffusion inside the pore. The former step is well studied in the literature. At elevated temperatures, above LCST, the polymer 48,49
M AN U
shows hydrophobic properties and hydrogen bonds between the drug and the polymer break . The latter step is somehow challenging. At elevated temperatures drug releases from the
polymer surface. At the same time, high temperature enhances drug diffusion within the porosity. However, this enhancement in diffusion coefficient is also valid for plain CMK. A probable explanation for drug release mechanism is desorption from the polymer surface as
TE D
a result of thermosensitivity followed by enhancement in diffusion induced by combined effect of high temperature and turbulence in the pore due to polymer shrinkage. This turbulence created convective mass transfer driving force and significantly enhances overall drug release. Based on this proposed mechanism, the release is mainly governed by
EP
thermosensitivity of the polymer; the more the volume of polymer, the more the release volume. Confirmed by nitrogen adsorption and SEM results for the CMK-P1 more pore
AC C
volume is occupied by the polymer. Consequently, effect of thermosensitivity and drug release behavior is more pronounced for CMK-P1. This is why the burst release, and consequently the overall release for CMK-P1 are reasonably higher than that of CMK-P4. Cytotoxicity
The cell viabilities from the MTT assays are shown in Figure 7 by incubating HEK 293T cells with CMK-P1 and CMK-P4 over a range of concentrations up to 2.5 mg/mL for 48 h. Results revealed that for CMK-P1, cell survival was approximately more than 80% over the test concentration range. However, the viability decreased slightly by increasing the concentration. Cell viability decreased from 80% at 0.3125 mg/mL to 60% at 2.5 mg/mL for
ACCEPTED MANUSCRIPT CMK-P4. These results suggest low in vitro cytotoxicity as it represents a much higher intravenous material dose than required for in vivo drug delivery 50 and comparable with the cytotoxicity of other nano- or micro carriers for drug delivery 51. Based on our previous work 39
, the lower cell viability in case of CMK-P4 is not due to the intrinsic toxicity of the
polymer. PNIPAAm-400n precipitates at 28 ˚C and the culture temperature is 37 ˚C which is
RI PT
9 ˚C higher than the polymer LCST. Polymer chain entanglements result in a barrier which covers the cells’ surface and it could get more dense and impermeable with a stronger entanglement and less pore sizes by continuing temperature rise above the LCST which subsequently hurdle nutrients and oxygen delivery to the cells.
SC
5- Conclusion
In summary, a properly-synthesized composite of OMC and thermosensitive polymer was
M AN U
shown to be a desirable carrier for drug delivery purposes. Incorporation of polymers in CMK was characterized by nitrogen adsorption isotherm and SEM, showing a uniform incorporation for PNIPAAm-100n, but a non-uniform one for PNIPAAm-400n. PNIPAAm400n had more residual on the external surface. Polymer-embedded samples showed less ultimate drug adsorption capacities, but significantly higher drug loading rates and drug
TE D
release capacities. The release capacity of the polymer-embedded samples was shown to be significantly improved with the temperature which is a result of polymer thermosensitivity. The polymer with the less chain length, PNIPAAm-100n, showed a smaller loading capacity compared to PNIPAAm-400n, but a reasonably higher release capacity. This behavior is
EP
attributed to the better pore filling of PNIPAAm-100n which results in a more effective release capacity. Cytotoxicity results confirm bio-compatibility of the synthesized materials
AC C
for possible future biological applications. Acknowledgements
Authors thank Dr. Moein Navvab Kashani and Dr. Hamideh Elekaei for sharing their facilities and for their assistance with the data analysis and interpretation. References 1. Gupta PK 1990. Drug targeting in cancer chemotherapy: a clinical perspective. Journal of pharmaceutical sciences 79(11):949-962. 2. Bae YH, Park K 2011. Targeted drug delivery to tumors: myths, reality and possibility. Journal of Controlled Release 153(3):198.
ACCEPTED MANUSCRIPT 3. Lu Y, Chen S 2004. Micro and nano-fabrication of biodegradable polymers for drug delivery. Advanced drug delivery reviews 56(11):1621-1633. 4. Qian KK, Bogner RH 2012. Application of mesoporous silicon dioxide and silicate in oral amorphous drug delivery systems. Journal of pharmaceutical sciences 101(2):444-463. 5. Nigmatullin R, Thomas P, Lukasiewicz B, Puthussery H, Roy I 2015. Polyhydroxyalkanoates, a family of natural polymers, and their applications in drug delivery. Journal of Chemical Technology and Biotechnology 90(7):1209-1221.
RI PT
6. Gupta P, Vermani K, Garg S 2002. Hydrogels: from controlled release to pHresponsive drug delivery. Drug discovery today 7(10):569-579. 7. Saylor DM, Kim CS, Patwardhan DV, Warren JA 2009. Modeling microstructure development and release kinetics in controlled drug release coatings. Journal of pharmaceutical sciences 98(1):169-186.
SC
8. Kim J-H, Kim Y-S, Kim S, Park JH, Kim K, Choi K, Chung H, Jeong SY, Park R-W, Kim I-S, Kwon IC 2006. Hydrophobically modified glycol chitosan nanoparticles as carriers for paclitaxel. Journal of Controlled Release 111(1–2):228-234.
M AN U
9. Wang Y, Zhao Q, Han N, Bai L, Li J, Liu J, Che E, Hu L, Zhang Q, Jiang T, Wang S 2015. Mesoporous silica nanoparticles in drug delivery and biomedical applications. Nanomedicine: Nanotechnology, Biology and Medicine 11(2):313-327. 10. Dong K, Liu Z, Li Z, Ren J, Qu X 2013. Hydrophobic Anticancer Drug Delivery by a 980 nm Laser-Driven Photothermal Vehicle for Efficient Synergistic Therapy of Cancer Cells In Vivo. Advanced Materials 25(32):4452-4458. 11. Zhao M-X, Zeng E-Z, Zhu B-J 2015. The Biological Applications of Inorganic Nanoparticle Drug Carriers. ChemNanoMat 1(2):82-91.
TE D
12. Ma Pa, Xiao H, Li C, Dai Y, Cheng Z, Hou Z, Lin J 2015. Inorganic nanocarriers for platinum drug delivery. Materials Today 18(10):554-564.
EP
13. Son SJ, Bai X, Lee SB 2007. Inorganic hollow nanoparticles and nanotubes in nanomedicine: Part 2: Imaging, diagnostic, and therapeutic applications. Drug Discovery Today 12(15–16):657-663.
AC C
14. Hu Y, Zhi Z, Wang T, Jiang T, Wang S 2011. Incorporation of indomethacin nanoparticles into 3-D ordered macroporous silica for enhanced dissolution and reduced gastric irritancy. Eur J Pharm Biopharm 79(3):544-551. 15. Ouyang Y, Shi HM, Fu RW, Wu DC 2013. Highly Monodisperse Microporous Polymeric and Carbonaceous Nanospheres with Multifunctional Properties. Sci Rep-Uk 3. 16. Swarnalatha S, Selvi PK, Ganesh Kumar A, Sekaran G 2008. Nanoemulsion drug delivery by ketene based polyester synthesized using electron rich carbon/silica composite surface. Colloids and Surfaces B: Biointerfaces 65(2):292-299. 17. Zhu W, Zhao Q, Zheng X, Zhang Z, Jiang T, Li Y, Wang S 2014. Mesoporous carbon as a carrier for celecoxib: The improved inhibition effect on MDA-MB-231 cells migration and invasion. Asian Journal of Pharmaceutical Sciences 9(2):82-91. 18. Zhu SM, Chen CX, Chen ZX, Liu XY, Li Y, Shi Y, Zhang D 2011. Thermoresponsive polymer-functionalized mesoporous carbon for controlled drug release. Mater Chem Phys 126(1-2):357-363.
ACCEPTED MANUSCRIPT 19. Kim T-W, Chung P-W, Slowing II, Tsunoda M, Yeung ES, Lin VSY 2008. Structurally Ordered Mesoporous Carbon Nanoparticles as Transmembrane Delivery Vehicle in Human Cancer Cells. Nano Letters 8(11):3724-3727. 20. Wu W, Wieckowski S, Pastorin G, Benincasa M, Klumpp C, Briand J-P, Gennaro R, Prato M, Bianco A 2005. Targeted Delivery of Amphotericin B to Cells by Using Functionalized Carbon Nanotubes. Angewandte Chemie International Edition 44(39):63586362.
RI PT
21. Pastorin G, Wu W, Wieckowski S, Briand J-P, Kostarelos K, Prato M, Bianco A 2006. Double functionalisation of carbon nanotubes for multimodal drug delivery. Chem Commun (11):1182-1184. 22. Bhirde AA, Patel V, Gavard J, Zhang G, Sousa AA, Masedunskas A, Leapman RD, Weigert R, Gutkind JS, Rusling JF 2009. Targeted Killing of Cancer Cells in Vivo and in Vitro with EGF-Directed Carbon Nanotube-Based Drug Delivery. ACS Nano 3(2):307-316.
SC
23. Mano JF 2008. Stimuli-Responsive Polymeric Systems for Biomedical Applications. Advanced Engineering Materials 10(6):515-527.
M AN U
24. Alarcon CdlH, Pennadam S, Alexander C 2005. Stimuli responsive polymers for biomedical applications. Chemical Society Reviews 34(3):276-285. 25. Kost J, Langer R 2001. Responsive polymeric delivery systems. Advanced Drug Delivery Reviews 46(1–3):125-148. 26. Qiu Y, Park K 2001. Environment-sensitive hydrogels for drug delivery. Advanced Drug Delivery Reviews 53(3):321-339. 27. Liu F, Urban MW 2010. Recent advances and challenges in designing stimuliresponsive polymers. Progress in Polymer Science 35(1–2):3-23.
TE D
28. Kjøniksen AL, Calejo MT, Zhu K, Cardoso AMS, de Lima MCP, Jurado AS, Nyström B, Sande SA 2014. Sustained Release of Naltrexone from Poly (N‐ Isopropylacrylamide) Microgels. Journal of pharmaceutical sciences 103(1):227-234.
EP
29. Wang Y, Xu H, Wang J, Ge L, Zhu J 2014. Development of a Thermally Responsive Nanogel Based on Chitosan–Poly (N‐Isopropylacrylamide‐co‐Acrylamide) for Paclitaxel Delivery. Journal of pharmaceutical sciences 103(7):2012-2021.
AC C
30. Rzaev ZMO, Dinçer S, Pişkin E 2007. Functional copolymers of Nisopropylacrylamide for bioengineering applications. Progress in Polymer Science 32(5):534-595. 31. Wang J, Yu X, Li Y, Liu Q 2007. Poly(3,4-ethylenedioxythiophene)/Mesoporous Carbon Composite. The Journal of Physical Chemistry C 111(49):18073-18077. 32. Wu S, Zhao X, Li Y, Du Q, Sun J, Wang Y, Wang X, Xia Y, Wang Z, Xia L 2013. Adsorption properties of doxorubicin hydrochloride onto graphene oxide: Equilibrium, kinetic and thermodynamic studies. Materials 6(5):2026-2042. 33. Yang X, Zhang X, Liu Z, Ma Y, Huang Y, Chen Y 2008. High-Efficiency Loading and Controlled Release of Doxorubicin Hydrochloride on Graphene Oxide. The Journal of Physical Chemistry C 112(45):17554-17558. 34. Shen H, Zhang L, Liu M, Zhang Z 2012. Biomedical applications of graphene. Theranostics 2(3):283-294.
ACCEPTED MANUSCRIPT 35. Zhang L, Xia J, Zhao Q, Liu L, Zhang Z 2010. Functional Graphene Oxide as a Nanocarrier for Controlled Loading and Targeted Delivery of Mixed Anticancer Drugs. Small 6(4):537-544. 36. Song E, Han W, Li C, Cheng D, Li L, Liu L, Zhu G, Song Y, Tan W 2014. Hyaluronic Acid-Decorated Graphene Oxide Nanohybrids as Nanocarriers for Targeted and pH-Responsive Anticancer Drug Delivery. ACS Applied Materials & Interfaces 6(15):11882-11890.
RI PT
37. Zhao D, Feng J, Huo Q, Melosh N, Fredrickson GH, Chmelka BF, Stucky GD 1998. Triblock Copolymer Syntheses of Mesoporous Silica with Periodic 50 to 300 Angstrom Pores. Science 279(5350):548-552.
SC
38. Mellati A, Kiamahalleh MV, Madani SH, Dai S, Bi J, Jin B, Zhang H 2016. Poly(Nisopropylacrylamide) hydrogel/chitosan scaffold hybrid for three-dimensional stem cell culture and cartilage tissue engineering. Journal of Biomedical Materials Research Part A 104(11):2764-2774.
M AN U
39. Mellati A, Valizadeh Kiamahalleh M, Dai S, Bi J, Jin B, Zhang H 2016. Influence of polymer molecular weight on the in vitro cytotoxicity of poly (N-isopropylacrylamide). Materials Science and Engineering: C 59:509-513. 40. Mellati A, Fan C-M, Tamayol A, Annabi N, Dai S, Bi J, Jin B, Xian C, Khademhosseini A, Zhang H 2016. Microengineered 3D cell-laden thermoresponsive hydrogels for mimicking cell morphology and orientation in cartilage tissue engineering. Biotechnology and Bioengineering:n/a-n/a. 41. Matyjaszewski K, Xia J 2001. Atom Transfer Radical Polymerization. Chemical Reviews 101(9):2921-2990.
TE D
42. Patten TE, Matyjaszewski K 1998. Atom Transfer Radical Polymerization and the Synthesis of Polymeric Materials. Advanced Materials 10(12):901-915. 43. Sing KSW, Everett DH, Haul RAW, Moscou L, Pierotti RA, Rouquerol J, Siemieniewska T 1985. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity. Pure Appl Chem 57(4):603.
EP
44. Brunauer S, Emmett PH, Teller E 1938. Adsorption of Gases in Multimolecular Layers. Journal of the American Chemical Society 60(2):309-319.
AC C
45. Sing KSW, Rouquerol F, Llewellyn P, Rouquerol J. 2014. 9 - Assessment of Microporosity. In Maurin FRRSWSL, editor Adsorption by Powders and Porous Solids (Second Edition), ed., Oxford: Academic Press. p 303-320. 46. Sing KSW, Rouquerol F, Rouquerol J, Llewellyn P. 2014. 8 - Assessment of Mesoporosity. In Maurin FRRSWSL, editor Adsorption by Powders and Porous Solids (Second Edition), ed., Oxford: Academic Press. p 269-302. 47. Neimark AV, Lin Y, Ravikovitch PI, Thommes M 2009. Quenched solid density functional theory and pore size analysis of micro-mesoporous carbons. Carbon 47(7):16171628. 48. Zhu S, Chen C, Chen Z, Liu X, Li Y, Shi Y, Zhang D 2011. Thermo-responsive polymer-functionalized mesoporous carbon for controlled drug release. Materials Chemistry and Physics 126(1–2):357-363.
ACCEPTED MANUSCRIPT 49. Deshmukh S, Mooney DA, McDermott T, Kulkarni S, Don MacElroy JM 2009. Molecular modeling of thermo-responsive hydrogels: observation of lower critical solution temperature. Soft Matter 5(7):1514-1521. 50. Chan JM, Zhang L, Yuet KP, Liao G, Rhee J-W, Langer R, Farokhzad OC 2009. PLGA–lecithin–PEG core–shell nanoparticles for controlled drug delivery. Biomaterials 30(8):1627-1634.
AC C
EP
TE D
M AN U
SC
RI PT
51. Horcajada P, Chalati T, Serre C, Gillet B, Sebrie C, Baati T, Eubank JF, Heurtaux D, Clayette P, Kreuz C, Chang J-S, Hwang YK, Marsaud V, Bories P-N, Cynober L, Gil S, Ferey G, Couvreur P, Gref R 2010. Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging. Nat Mater 9(2):172-178.
ACCEPTED MANUSCRIPT
Table 1. Structural properties of plain CMK and polymer-embedded CMK samples: CMK-P4 and CMK-P1 Micropore
Total pore
Mesopore
surface area*
volume**
volume***
volume fraction
(cm2/g)
(cm3/g)
(cm3/g)
(%)
CMK
1055
0.34
1.41
0.76
CMK-P4
637
0.21
0.75
0.72
CMK-P1
489
0.15
SC
Sample ID
RI PT
BET equivalent
0.63
AC C
EP
TE D
M AN U
* Surface area is calculated based on BET method 44. **Micropore volume is calculated based on DR method 45. ***Total pore volume is calculated based on liquid volume adsorbed at p/po≈1 46.
0.76
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT