Programmable delivery of hydrophilic drug using dually responsive hydrogel cages

Journal of Controlled Release 117 (2007) 396 – 402 www.elsevier.com/locate/jconrel

Programmable delivery of hydrophilic drug using dually responsive hydrogel cages Jingxia Gu a , Fan Xia b , Yan Wu b,⁎, Xiaozhong Qu a,⁎, Zhenzhong Yang a , Lei Jiang b a

State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100080, China 1 b National Center for Nanoscience and Technology, Beijing 100080, China 1 Received 31 August 2006; accepted 28 November 2006 Available online 8 December 2006

Abstract Micro-capsules normally encapsulate therapeutic agents only inside their cavities. In this paper, we report on the synthesis of dually responsive poly(N-isopropylacrylamide) (PNiPAM)-co-acrylic acid (AA) hydrogel cages sub-micrometer in size and the use of these cages as drug carriers. The cavity structure of the cages can enhance volume phase transition compared to solid gel particles, thus favoring drug loading and release. TEM images and FT-IR spectra confirmed that the model drug isoniazid (INH) is located in two regions: within the shell and inside the cavity of the cages. The drugs residing in the shell can form hydrogen bonds with the cage matrix, while the drugs in the cavity are interaction free with the carrier. This difference from the residency of drugs exploited to a structure induced drug release which was programmable controlled by external pH and temperature. In vitro drug release studies showed that in a neutral medium (pH = 7.4), major drugs were preserved within the shell, while in an acidic medium (pH = 1.2), nearly all of the drugs were released due to the dissociation of hydrogen bonds. © 2006 Elsevier B.V. All rights reserved. Keywords: Polymeric hydrogel; Cages; Responsive; Programmable; Drug delivery

1. Introduction Sub-micron sized polymeric spheres are promising delivery systems in the pharmaceutical field. They are ideal carriers for low molecular weight drugs, oligonucleotides and peptides because they can increase the solubility of hydrophobic drugs, reduce toxicity and enhance availability of those therapeutic agents [1–7]. Studies have shown that the spheres can interact strongly with the gastrointestinal mucuosa and cellar linings, thus facilitating the drug uptake when administrated orally. They also possess slow clearance from blood and can penetrate into small capillaries allowing for efficient drug accumulation and further exploitability of target drug delivery. Compared to rigid particles such as polyester spheres, hydrogel spheres display enhanced drug loading capacity and better dispersibility in aqueous media [1,8]. The inclusion of stimuli responsive polymers in the hydrogel spheres enabled ⁎ Corresponding authors. Tel.: +86 10 82619206; fax: +86 10 62559373. E-mail addresses: [email protected] (Y. Wu), [email protected] (X. Qu). 1 These institutions contributed equally to this work. 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.11.029

switchable “on–off” release of drugs and biomolecules in response to external stimuli [9–12]. The typical response of hydrogel spheres is in micro-second, much faster than their corresponding bulk gels, which is conducive to the controlled release of drugs [13,14]. Among the responsive hydrogel spheres, poly (N-isopropylacrylamide) (PNiPAM) spheres have been extensively investigated as site-specific delivery systems [15–20]. Carboxyl groups are commonly incorporated into the PNiPAM hydrogel matrix to modify the swelling behavior, resulting in the formation of hydrogels which are dually responsive to both pH and temperature. The programmed drug release from those hydrogels can be fine-tuned by changing the pH or/and temperature of the external environment [14,21–25]. In this paper, we report on the synthesis of poly(Nisopropylacrylamide)-co-acrylic acid (PNiPAM/AA) copolymer hydrogel hollow cages and the release of a model hydrophilic drug for this system. To date, crosslinked micelles [26,27] and hollow spheres synthesized through layer-by-layer technique [28] have been developed to encapsulate pharmaceuticals. Although hollow spheres with temperature responsive hydrogel shells have been synthesized [29,30], there are few

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reports on the investigations of their drug encapsulation and release properties. In this work, isoniazid (INH), an antitubercular drug was chosen as the model drug. INH has an isoelectric point of 1.82 and an octanol–water partition coefficient (log P) of − 0.72 indicating high hydrophilicity of the drug. Liposomes, polyester and polysaccharide particles have been used as sustained colloidal delivery agents for INH [31–33]. Different from the previous reports, we will focus on the use of PNiPAM/ AA hydrogel hollow cages to deliver a hydrophilic drug. It is well known that drug release kinetics is greatly influenced by the interactions between drug and polymeric carriers [1,31,34]. For hydrogel cages described in this paper, the drug is located in two regions: within the gel shell and inside the cavity of the hollow hydrogel cages. Thus the interactions between the drug and polymer matrix in these two regions are different. It is therefore expected that a programmable drug release induced by the structure of the carrier can be achieved by externally controlling the release kinetics of those drugs with different interactions. 2. Experimental Section 2.1. Materials N-isopropylacrylamide (NiPAM), N,N′-methylene bis(acrylamide) (BIS), isoniazid (INH) and phosphate buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, USA). Tetraethyoxysilane (TEOS), acrylic acid (AA), 3-(trimethoxysily) propylmethacrylate (MPS), hydrofluoric acid (HF), sodium dodecyl sulfate (SDS) and ammonium persulfate (APS) were obtained from Shanghai Chemical Reagents Company (Shanghai, China). NiPAM was recrystallized from n-hexanes and dried in vacuum prior to use. All the other reagents were used as received. 2.2. Synthesis PNiPAM/AA hydrogel cages 2.2.1. Synthesis of SiO2 template colloidal particles A typical synthesis procedure was depicted as follow: 13 g of TEOS was injected into a stirring solution containing 125 g of ethanol, 25 g of ammonia and 50 g of water. After the mixture was stirred for 24 h, an excessive amount of MPS (2.0 g) was added to introduce vinyl groups onto the silica particle surface [35]. Residual MPS was removed by washing the particles with ethanol and water. The MPS-modified silica particles were then re-dispersed in water at a solid content of 1.0 wt.% for further use. The average diameter of the colloidal silica particles was determined to be approximately 300 nm from the TEM images. 2.2.2. Synthesis of PNiPAM/AA hydrogel cages An appropriate amount of NiPAM, AA and BIS was dissolved in 120 mL of water containing 0.042 g of SDS. The solution was then added into 41 g of 1.0 wt.% silica aqueous dispersion. The monomers/SiO2 weight ratio was fixed at 3:1. The dispersion was purged with nitrogen for 40 min before being heated to 70 °C. APS (0.053 g) dissolved in 3 mL of water was injected to initiate the polymerization. The reaction mixture

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was stirred for 6 h at 70 ± 1 °C under the nitrogen atmosphere. The resulting SiO2-PNiPAM/AA core-shell particles were centrifuged and thoroughly washed with water and methanol to remove SDS and unreacted monomers [36]. The purified SiO2-PNiPAM/AA core-shell particles were immersed in 20 mL of 10% HF aqueous solution for 4 h to etch the silica cores and the solution was then dialyzed against water until the pH became neutral. The dialysate was freeze-dried to give PNiPAM/AA cages as white powder. 2.3. Characterization 2.3.1. Dynamic light scattering The average hydrodynamic radius of PNiPAM/AA cages at different temperatures and pH were determined using a laser light scattering spectrometer (ALV Co., Germany) with an ALV-5000 digital time correlator and a Helium–Neon laser (Model 127, output power of 40 mW with the wavelength at 632.8 nm). The sample cell was equipped with a water bath with temperature accuracy of ± 0.1 °C. The measurements were carried out at a fixed angle of 30°. The Laplace inversion and accumulant analysis of the intensity-intensity time correlation function were performed to obtain the translational diffusion coefficient distribution G(D) and the hydrodynamic radius distribution f(Rh). The average hydrodynamic radius was then calculated using the Stokes–Einstein equation. 2.3.2. Transmission electron microscopy (TEM) TEM images were obtained using a Hitachi H-600 transmission electron microscope operating at 250 kV. Samples were deposited onto carbon coated copper grids, dried at room temperature and were stained with 1 wt.% phosphate–tungstic acid (PTA) aqueous solution when necessary. 2.3.3. Infrared spectroscopy (FT-IR) Infrared spectroscopy was performed on a Brukerequinox 55 FT-IR spectrometer. Freeze-dried cages before and after being loaded with INH were pressed with KBr under vacuum. For each sample, 100 scans were recorded from 4000 to 400 cm- 1 with a resolution of 2 cm− 1. 2.4. Drug loading into PNiPAM/AA cages INH was dissolved in distilled water to produce 4 mg/mL and 20 mg/mL solutions. An appropriate volume of the INH solution was pipetted into a test tube containing 5 mg of freezedried PNiPAM/AA cages. The volume of the INH solution was calculated to obtain a drug: cage ratio of 1:1 (w/w). The hydrogel cages were completely immersed in the INH solution for 24 h at room temperature to allow an equilibrium loading. Then the dispersion was centrifuged at 6000 rpm for 5 min to separate the loaded cages. The amount of drugs loaded in the cages was determined by measuring the amount of drug in the loading solution and in the supernatant using a UV spectrophotometer with the detection wavelength of 262 nm. The detection limit was 1 μg/mL with a linear standard absorbance– concentration curve over the range 1–25 μg/mL.

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Table 1 Recipe of PNiPAM/AA hydrogel cages and their swelling behavior Sample

NiPAM (mol%)

AA (mol%)

BIS (mol%)

NiPAM/AA (molar ratio)

pH for DLS

Hydrodynamic diameter at 25 °C (RH,25°C) (nm)

Hydrodynamic diameter at 37 °C (RH,37 °C) (nm)

LCST ( °C)

Swell ratio a

1 2 3 4

81 81 88 63

9 9 10 27

10 10 2 10

9:1 9:1 9:1 7:3

4.0 6.5 6.5 4.0

309 322 367 306

265 286 240 198

35 33 36 32

1.59 1.42 3.58 3.69

a

Swell ratio is defined as (RH,25°C/RH,37 °C)3 [29]. Double distilled water was used as solvent.

2.5. In vitro drug release from the PNiPAM/AA cages Drug loaded cages were re-dispersed in either 2 mL of PBS (pH 7.4) or simulated gastric fluid (0.1M HCl/34 mM NaCl, pH 1.2) immediately after the loading process. The dispersion was then transferred into a dialysis bag (molecular weight cut off 12 kDa) and the bag was subsequently placed in a 25 mL flask containing 15 mL of PBS or simulated gastric fluid. 1 mL of solution was collected from the flask periodically and the released drug was determined spectrophotometrically. The volume of the release medium in the flask was kept constant by adding 1 mL of fresh medium after each sampling. All drug release data were averaged with three measurements. In a separate experiment, two-step release of INH was carried out by dialyzing the drug-loaded cages firstly in PBS (15 mL, pH 7.4) at either 25 °C or 37 °C for 110 min and subsequently in the simulated gastric fluid (15 mL, pH 1.2) at 37 °C. The sampling methods followed the same procedure as mentioned above.

size and shell thickness of the cage can be synthetically controlled by using silica template core of varied size and different amount of monomers. It was shown that the cages were both temperature and pH responsive. LCST of the cages was affected by the NiPAM/AA ratio, the extent of crosslinking and the change of pH. The cages had a larger size in a neutral aqueous environment than in an

3. Results and discussion 3.1. Characterization of PNiPAM/PAA cages The PNiPAM/PAA hydrogel cages were prepared by SiO2templated polymerization followed by the removal of the template core. The silica core was synthesized via Stöber sol– gel process, which was found monodisperse with an average diameter of 300 nm (TEM). 3-(trimethoxysily) propyl methacrylate (MPS) was used to modify the core surface by introducing vinyl groups. SiO2-PNiPAM/AA core-shell particles were polymerized using N,N′-methylene bis(acrylamide) (BIS) as a crosslinker. The monomer feeding ratios are listed in Table 1. The inclusion of double bonds on the SiO2 surface and the addition of SDS could avoid the secondary nucleation during the polymerization. The resulting composite particles were also monodisperse in size as shown in Fig. 1A. PNiPAM/ PAA hydrogel cages were obtained by chemically etching the silica cores with HF. FT-IR spectra indicated that the polymers were stable in the presence of HF. Core-shell structure of the composite particles could be distinguished (Fig. 1A, B), especially from those particles of which the silica cores were partially removed (Fig. 1B). TGA result indicated that the residual SiO2 was 16 wt.% (Fig. 1B). After the silica was completely removed with prolonged etching time (4 h) confirmed by FT-IR and TGA analysis, hydrogel cages were gained with their spherical contour well retained (Fig. 1C). The

Fig. 1. TEM images of SiO2-PNiPAM/AA core-shell particles (A), PNiPAM/AA hydrogel cages with SiO2 cores partially removed (etched for 2 h in 10% HF aqueous solution) (B) and PNiPAM/AA hydrogel cages with SiO2 cores completely removed (etched for 4 h in 10% HF aqueous solution) (C).

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acidic medium with pH lower than the pKa of PAA (pKa = 4.6). The volume phase transition of the cages with a high crosslinking level, i.e. 10 mol% of the total monomer content, was reversible during the heating and cooling cycle (Fig. 2). With the increase in AA content, the decrease in crosslinking level or the decrease of pH, the collapse of the cages over LCST was accelerated and resulted in a larger swell ratio (Fig. 2 and Table 1). Compared to a solid PNiPAM/AA hydrogel sphere, the size of the hydrogel cage with the same composition changed from 367 nm to 200 nm over the transition temperature, while the former changed from 320 nm to 210 nm [25]. Thus, the cavity structure could bring a more profound volume transition, which is conducive to drug loading and delivery. 3.2. Drug loading PNiPAM/AA (9:1 molar ratio) hydrogel cages with crosslinking level of 10 mol% were used to study the drug loading and release. INH was loaded at 25 °C and neutral pH. With INH loading concentration increased from 4 mg/mL to 20 mg/mL, the amount of encapsulated drugs increased from 118 ± 12 mg/g to 495 ± 38 mg/g of hydrogel cages. Since the drug loading capacity is influenced by the swelling degree and the mesh size of a hydrogel [37,38], the cavity of the hydrogel cages favors the drug loading compared to solid hydrogel spheres. The drug location within the cage was investigated by TEM. As shown in Fig. 3A, without negative staining, the shape of drug loaded cages remained in spherical when dispersed in water, indicating an incursion of drugs [39], because in comparison, the cages before drug loading were hardly observed by TEM if not being negatively stained. We therefore postulate that the drug locates in the hydrogel shell and within the cavity. To confirm this hypothesis, drug loaded particles were re-dispersed in ethanol to induce shell contraction of the cages. Since INH is insoluble in ethanol, very few drug molecules within the cavity will be released into the external environment while the drugs in the shell will be forced to release. In Fig. 3B, the drug loaded cages displayed crystal like structures rather than spherical shape. The size was approximately 120–150 nm, much smaller than those shown in Fig. 3A. After being negatively stained, the gel shell

Fig. 2. Hydrodynamic diameter of PNiPAM/AA hydrogel cages (NiPAM/ AA = 9:1 molar ratio) in water at various pH as a function of temperature.

Fig. 3. TEM images of drug loaded PNiPAM/AA cages. The cages were dispersed in distilled water (A), and in ethanol (B).

could be clearly distinguished with a thickness of 20 nm. This can be explained by the dehydration of the gel shell in the presence of ethanol and the crystallization of the INH within the cavity due to outward diffusion of water. 3.3. Drug release Fig. 4 shows the cumulative drug release from the cages at various temperatures and pH. The drug release rate was faster at a higher temperature, i.e. 37 °C than at a low temperature, i.e. 25 °C. This was caused by the collapse of the hydrogel shell at 37 °C. As INH is a small molecule and highly soluble in water (log P = − 0.72), the overall drug release rate is rapid at all release temperatures [37]. While the cumulative drug release in an acidic simulated gastric fluid (pH = 1.2) reached ca. 96% after 150 min, only 45%

Fig. 4. Release profiles of INH from PNiPAM/AA cages in PBS (circles) and simulated gastric fluid (squares) at various temperatures, i.e. 25 °C (solid symbols) and 37 °C (open symbols). The drug loading concentration was 20 mg/mL.

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Fig. 5. FT-IR spectra of empty PNiPAM/AA cages (a), INH (b), PNiPAM/AA cages loaded with 12% w/w (c) and 40% w/w (d) of INH.

was released in PBS (pH = 7.4). This phenomenon cannot be explained by the mechanisms of squeezing out effect and the acid–base interaction between drug and PAA groups [24,40,41]. In our regime, IHN has an isoelectric point of 1.82 thus its net charge is zero at neural pH. It is unlikely that INH forms electrostatic interaction with the deprotonated PAA in the gel in a neutral medium. The major interaction between the matrix and INH at neutral pH is attributed to hydrogen bonding. FT-IR characterization confirmed this conjecture. After the cage was loaded with 40% w/w of INH, the absorption bands of carbonyl and N–H groups of the NiPAM shifted to higher wavenumbers centered at 1652 and 1553 cm− 1 from their original positions at 1649 and 1543 cm− 1 (Fig. 5a, d). INH can form strong inter-molecular hydrogen bonds indicated by a strong N–H stretching at 3110 cm− 1 (Fig. 5b). This peak became weak and shifted to 3115 cm− 1 after the drug was encapsulated, accompanied by a slight shift of the amine group of NiPAM at 3306 cm− 1. Meanwhile the INH absorption bands at 1666, 1635 and 1556 cm− 1 shifted to lower wavenumbers (Fig. 5b–d). The changes of these characteristic peaks became more pronounced with the increase of drug loading, confirming the formation of hydrogen bonds between the drugs and the hydrogel matrix (Fig. 5c–d). With a higher drug loading, the characteristic peak of carbonyl stretching of INH at 1666 cm− 1 became visible, suggesting the existence of free drugs within the cavity (Fig. 5d). Therefore, when the cages were in PBS, although the presence of salts might weaken the interaction, certain amount of drugs would remain in hydrogen bonding with the NiPAM segments of the shell and hence these drugs would be preserved in the matrix. At pH 1.2, the drug molecules were protonated and unable to form strong hydrogen bonds like at pH 7.4 with the gel matrix. Additionally, carboxyl groups in the matrix were fully

protonated to give –COOH, which could form hydrogen bonds with PNiPAM [42]. The two factors caused the dissociation of hydrogen bonds between the drug and the matrix and accelerated the complete drug release in the simulated gastric fluid. The effect of drug concentration in pH sensitive hydrogels on the cumulative release of hydrophilic drugs has been extensively investigated [43,44]. The cumulative release of gentamincin and cefatroxil is augmented with the increase of drug loading. This is due to the increase of free/unbound drugs within the polymer networks and a favorable diffusion of the drugs when loaded with higher levels. However, we found that the cumulative release of INH from the hydrogel cages at pH 7.4 did not change drastically when the drug loading was increased from 11.8% w/w (ca. 50% released) to 49.5% w/w (ca. 45% released, Fig. 4), but could increase with the decrease in shell thickness of the cages. This result suggests a new mechanism: structure induced delivery. Since major amount of free INH molecules were located inside the cavity, although there were unbound drugs in the shell, the ratio of bound drugs to the free drugs would be still determined by the portions of drugs located in gel shell to those in the cavity. Guided by the “structure induced drug delivery”, Fig. 6 illustrates a programmable two-step drug release from the hydrogel cages. INH was loaded into the hydrogel cages at room temperature and neutral pH. The drug molecules existed both in the shell and inside the cavity, either with hydrogen bonding to the PNiPAM or having no interaction with the matrix. At the first stage, in a neutral medium the release of unbound drugs at interface of the cages resulted in the transfer of the free drugs from inside the cavity to the gel shell and further to the interface to compensate the less of released drugs. This is because the hydrogen bonding between the drug and the polymer led to a higher drug concentration at the interface. The release would stop when there was no free drug inside the cages. At the second stage, in an acidic medium the drugs remaining bounded in the shell were released due to the dissociation of the drug–matrix interaction. It is noticeable that the drug release at the second stage (37 °C in acidic medium) is influenced by the thermo history of the former external condition. Having been incubated at 37 °C for 2 h, the shell-entrapped drugs could not be liberated after the neutral medium was changed to the acidic medium

Fig. 6. Structure induced two-step release of INH from PNiPAM/AA cages.

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(Fig. 6). A possible explanation is that a long storage at a temperature above LCST induced coil-to-globule transition of the PNiPAM networks, causing some of the drugs in the shell being well trapped [45]. Furthermore the cages would remain in globule conformation in the second stage (37 °C). However, if the hydrogel cages are incubated at 25 °C in the first stage, the conformation of cages was kept in a swollen state, which resulted in a higher level of drug release at the second stage.

[9] [10]

[11]

4. Conclusions [12]

Temperature and pH dually responsive hydrogel cages with hollow cavities have been synthesized using SiO2-template emulsion copolymerization method, which was demonstrated to be an efficient way to prepare sub-micrometer size hollow spheres for the entrapment of useful chemicals. A model hydrophilic drug, INH, was loaded into the cages and found to be located in both the shell and the cavity. The drug residing in the shell forms hydrogen bonds with the polymer matrix and drug in the cavity remains unbound. The unique characteristics of the hollow hydrogel cages could induce novel release behavior of the carried drugs, e.g. a programmable drug release in response to external stimuli. Acknowledgements The authors are grateful to Prof. Deyan Shen, Institute of Chemistry, for the FT-IR analysis. This work was financially supported by the State Key Project for Fundamental Research (2003CB716900), National Center for Nanoscience and Technology of China. XQ thanks the State Key Laboratory of Polymer Physics and Chemistry and Center for Molecules, Institute of Chemistry, Chinese Academy of Sciences for the Startup Funding. References

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