Vol.:(0123456789) Chemical Papers (2024) 78:4007–4018 https://doi.org/10.1007/s11696-024-03371-z ORIGINAL PAPER Designing of drug imprinted polymeric microcryogels for controlled release of Darunavir İsmet Şafak1 · Merve Çalışır1 · Monireh Bakhshpour‑Yucel2 · Necdet Sağlam3 · Adil Denizli1 Received: 22 September 2023 / Accepted: 14 February 2024 / Published online: 13 March 2024 © The Author(s), under exclusive licence to the Institute of Chemistry, Slovak Academy of Sciences 2024 Abstract Darunavir (DRV) is a crucial antiretroviral drug specifically developed for treating infections that require prolonged treat- ment. It has gained significant recognition as one of the top choices for combating AIDS, a condition caused by the human immunodeficiency virus. Biopolymeric materials like microcryogels become the center of attention in most research areas such as controlled release systems. These systems offer the advantage of precise drug administration, ensuring effective therapeutic outcomes through the delivery of specific drug doses. Microcryogels, characterized by their super macroporous, elastic, and spongy morphology, have emerged as a focal point in biomedical applications, particularly when combined with molecularly imprinted polymers. In this study, the controlled release and kinetics studies of the DRV were investigated with the DRV-imprinted poly(2-hydroxyethyl methacrylate) (pHEMA)-based microcryogels. Darunavir imprinted pHEMA microcryogels with different cross-linker ratios and different loaded drugs were prepared for studies of in vitro release of DRV; scanning electron microscopy, Brunauer–Emmett–Teller, and Fourier transform infrared spectroscopy methods have been considered suitable for the characterization of cryogels that have been designed and whose sensitivity has been enhanced by molecular imprinting. Cytotoxicity of DRV-imprinted microcryogels was also inspected using mouse fibroblast cell line L929. The comprehensive analysis results underscore the potential of these meticulously designed microcryogels, show- casing their utility in medical applications. Notably, these microcryogels exhibited controlled drug release, with efficiency levels of up to 85% and sustained release duration of 40 h, positioning them as a valuable option for advanced drug delivery systems in the medical field. Keywords DRV · Antiretroviral drug · HIV · pHEMA-based microcryogels · Controlled drug release systems Introduction The development of antiretroviral drugs has been signifi- cantly influenced by the emergence of acquired immuno- deficiency syndrome (AIDS). From the mid-1990s to the present, advancements in HIV management have trans- formed the landscape of HIV infection into a controlla- ble chronic condition. This transformation is attributed to early diagnosis, regular monitoring, and the introduction of antiretroviral drug therapies (Youle 2008). Over the years, substantial progress has been made in the development of antiretroviral drugs, resulting in the creation of effective, user-friendly, and highly potent protease inhibitors (PI)s. Approaches to treating HIV-1 protease (PR) inhibitors aim to provide patients with an extended and enhanced quality of life (Meyer et al. 2005). The mature human immunodefi- ciency virus HIV-1 protease (PR) is one of the most impor- tant enzymes in DRV drug discovery. Darunavir, introduced as the tenth-ranked member of the aspartyl protease cate- gory, stands out as a formidable tool created to combat AIDS (Li et al. 2014). On the other hand, DRV is the second- generation non-peptidic peptidomimetic PI and antiretrovi- ral drug that requires a high therapeutic dose, to deal with the problems such as severe side effects and drug toxicities (Tegeli et al. 2021; Mantena et al. 2015). Darunavir is used orally with ritonavir and other medicated combinations to * Adil Denizli denizli@hacettepe.edu.tr 1 Department of Chemistry, Hacettepe University, Ankara, Turkey 2 Department of Chemistry, Bursa Uludag University, Bursa, Turkey 3 Institute of Science and Engineering, Division of Nanotechnology and Nanomedicine, Hacettepe University, Ankara, Turkey http://crossmark.crossref.org/dialog/?doi=10.1007/s11696-024-03371-z&domain=pdf 4008 Chemical Papers (2024) 78:4007–4018 treat and prevent HIV/AIDS (Fenton and Perry 2007). In recent years, managing the drug dosage is the main focus of treatments for many reasons (Forbes et al. 1999). During the treatment, researchers conduct various studies to prolong the effectiveness of the drug for patients and minimize its side effects (Denizli et al. 1988). Controlled drug release systems are at the forefront of these studies and offer an effective treatment method based on delivering drugs at desired dos- ing intervals (Singh and Kim 2000). In traditional methods, as the drug is mixed into the blood, the level of active sub- stance in the blood should be kept within a range called the plasma concentration (therapeutic window) of a therapeutic agent effectively. This interval ranges from the minimum effective concentration to the minimum toxic concentration. Drug release systems focus on these two main concentration points, and the drug can be released over a long period in the therapeutic window range (Çetin and Denizli 2019). Drug release systems are also suitable for incorporating many innovative methods such as molecular imprinted polymers. Molecular imprinting technique has proved its effective- ness in several medical applications as a highly versatile approach. For instance, molecular imprinting is commonly applied to detect allergens or proteins at low levels, boost sensitivity in biosensor research, and ensure strong selectiv- ity in various detections. The technique is based on prepar- ing a template cavity for the desired molecule to increase molecular affinity (Byrne and Salian 2008). The high affinity is achieved through the use of specific building blocks and a cross-linking agent in the polymerization process (Spivak 2005). In this process, polymer matrices with special recog- nition and catalytic site properties are also obtained. When the molecule is removed from the polymer, target-specific cavities are occurred (Piletsky et al. 2001; Çetin and Denizli 2018). Molecular imprinted polymers exhibit high stability in pH changes, the presence of organic solvents, and high- temperature pressure environments. Therefore, it can easily adapt to the potential changes of the human body, so it can be applied as an effective treatment tool in drug release systems (Bakhshpour et al. 2018; Wu et al. 2020; Küp et al. 2020; Deng et al. 2021; Ganguly et al. 2018; Ueda et al. 1994). The involvement of cryogel structures in the proven effective- ness of molecular imprinting in drug release systems carries the studies to another point. Hydrogels are considered as a three-dimensional cross-linked natural or synthetic polymer network capable of absorbing large amounts of water or bio- logical fluid, and when this polymer network is formed at low temperatures, the structures formed are called cryogels. These functioning materials are classified as next-generation adsorbents thanks to their unique structural properties and flow dynamics of solutes of all sizes, ranging in size from 10 to 100 μm (Li et al. 2019; Aydin et al. 2017; El-Naggar et al. 2017; Radhouani et al. 2019; Lee et al. 2020). These adapt- able structures, demonstrated as beneficial in biomedical studies, distinguish themselves through their resilience to chemical and mechanical deformations. They serve as carriers for immobilizing antibodies, enzymes, and cells, and function as bioaffinity materials and gel bases for drug release systems with swelling kinetics (Ipate et al. 2018; Chambre et al. 2020; Koshy et al. 2018; Çetin and Denizli 2022; Dursun et al. 2016). The objective of this research is to present an effective and innovative method to the literature by combining molecular imprinting and cryogel approaches. Cryogels incorporating DRV through chelation with copper were synthesized, with N-Methacryloyl (L)-histidine methyl ester (MAH) serving as a co-functional monomer in imprint- ing within the structure. The release behaviors of DRV were observed in different loading concentrations, temperatures, and pH environments. In addition, in order to reach the most stable form of the cryogel structure, different cross-linker ratio cryogels were synthesized. Characterizations of the designed cryogels were made, and the results were shared. Experimental Materials Darunavir was obtained from Hacettepe University Hospi- tal Infectious Diseases and Clinical Microbiology Depart- ment. N-Methacryloyl-(L)-histidine methyl ester (MAH) monomer was purchased from NanoReg (Ankara, Turkey). Ammonium persulfate (APS), 2-hydroxyethyl methacrylate (HEMA), N,N,Nʹ ,Nʹ-tetramethylene diamine (TEMED), and methylene bisacrylamide (MBAAm) were obtained from Sigma Chemical Co. (St. Louis, MO). Deionized water (DW) was obtained from Thermo Scientific™ Barnstead™ Nanopure water purification device. Remained chemicals were acquired from Merck AG (Darmstadt, Germany). Preparation of pre‑complex used for molecular imprinted The investigation into the impact of chelated Cu(II) levels involved the synthesis of microcryogels containing three distinct Cu(II) concentrations. In the process of creating the MAH-Cu(II) pre-complex, the MAH functional monomer remained constant, while Cu(II) metal ions were incorpo- rated in molar ratios of 1:1, 1:2, and 1:3. These pre-com- plexes were derived from Cu(NO3)2·5H2O, resulting in pre- complex solutions of 1:1, 1:2, and 1:3. Following this, 1.0 mol of DRV was introduced into each of the pre-complex solutions. The ensuing mixture of MAH- Cu(II) and DRV was then dispersed in 1.0 mL of 10 mM phosphate buffer (PBS) with a pH of 7.4. Subsequently, the solution underwent thorough mixing on a rotator at 25 °C for a duration of 30 min. 4009Chemical Papers (2024) 78:4007–4018 Preparation of DRV‑imprinted microcryogels The synthesis process unfolded as follows: Initially, 1.3 mL of HEMA monomer was introduced into a beaker. Subse- quently, 0.283 g of MBAAm was dissolved in distilled water (DW) and then mixed with the HEMA solution. The total solution volume was adjusted to 12 mL. Next, the MAH- Cu(II)–DRV complex, previously prepared, was incorpo- rated into this mixture and stirred for 10 min. To initiate polymerization, TEMED (25 μL) and APS (20 mg) initiator pairs were swiftly added to the mixture. This resulting blend was carefully introduced into a mold featuring holes with dimensions of 200 μm in diameter and 500 μm in thickness. Polymerization commenced and continued for 24 h at a temperature of −14 °C, positioned between two glass plates. Following this phase, the micros- tencil mold was subjected to a lyophilization process for 2 h at −56 °C and 0.0010 mbar. Upon completion of this period, the DRV-imprinted (MIP) microcryogels formed between the glass plates were extracted from the microstencil mold after reaching room temperature. To eliminate any residual unreacted monomers, the cryogels were subjected to multi- ple washes with distilled water. The synthesis steps for microcryogels are visually rep- resented in Fig. 1. The same protocol was applied to create DRV non-imprinted (NIP) microcryogels, with the distinc- tion that DRV drug was omitted from the monomer phase during microcryogel formation. Fig. 1 Schematic representa- tion of injectable microcryogels synthesis 4010 Chemical Papers (2024) 78:4007–4018 For all complexes containing different Cu(II) ratios, this methodology was employed. Experimental results dem- onstrated a more consistent release pattern at a 1:1 ratio; consequently, further studies were conducted at this ratio. In the synthesis of NIP microcryogels, the MAH-Cu(II) pre- complex was utilized in a 1:1 mol ratio. Additionally, in the synthesis of pHEMA microcryogels without employing MAH-Cu (II) and DRV as target molecules, HEMA served as the monomer, and MBAAm functioned as the cross-linker in both MIP and NIP microcryogel production. Subsequently, the APS initiator was combined with TEMED and introduced into a microstencil mold with iden- tical dimensions. As part of this study, we embarked on the synthesis of MIP microcryogels, which featured varying pro- portions of MAH-Cu(II) and different ratios of cross-linker. Notably, the quantity of cross-linker played a critical role as one of the parameters influencing the swelling rate, surface area, and release kinetics within the microcryogels. It is worth noting that MBAAm, serving as the cross- linker, exhibits hydrophobic characteristics. Consequently, an escalation in the MBAAm ratio results in the augmenta- tion of less hydrophilic groups within the polymer struc- ture. Consequently, this augmentation leads to the creation of more robust and enduring polymer networks within the microcryogels. Characterization of microcryogels FTIR analysis The bulk structure of DRV-imprinted (MIP), non-imprinted (NIP), and pHEMA microcryogels was examined by FTIR. Before analysis, the microcryogels were dried by lyophili- zation for 24 h at −60 °C. Then, 2.0 mg of polymer sample was mixed with 98 mg of KBr and pounded in a mortar to obtain FTIR spectra. It was then turned into a fine pellet at a pressure of 600 kg/cm2 in a hydraulic press, and charac- terization of the material was performed in the wavelength range of 4600–400  cm−1. Surface morphology Surface morphologies of injectable microcryogels were investigated by scanning electron microscopy (SEM, GAIA3, Tescan, Czech Republic). In the first step, the microcryogels were lyophilized at −50 °C (0.050 mbar). Afterward, the samples were coated with a gold–palladium mixture (40:60) in a vacuum to make their surfaces conduc- tive. Subsequently, the surface morphology was observed in the SEM at various magnifications, and images were captured. Swelling tests In order to determine the swelling properties of the cryo- gels, the dry and swollen weights of the cryogels were taken separately. Dry cryogels, whose weights were meas- ured, were first soaked in water for two hours, and excess water was removed with absorbent paper and the residue was weighed. Equation 1 is used for determining the swell- ing rate Surface area measurements The cryogel disks' specific surface area in a dry state was determined utilizing a multipoint Brunauer–Emmett–Teller (BET) apparatus (Quantachrome, NOVA 2000, USA). Ini- tially, the cryogel disks were weighed, placed in a sample holder, and subjected to degassing with N2 gas at 150 °C for 1 h. Subsequently, the cryogels were re-weighed. Gas adsorption occurred at − 210 °C, followed by desorption at room temperature. The values obtained during the des- orption step were employed for calculating the specific surface area. Investigation of release behavior of Darunavir from MIP microcryogels The examination of the drug release characteristics of MIP microcryogels took place within an environment compris- ing a phosphate-buffered saline (PBS) solution with a pH of 7.4, maintained at a temperature of 37 °C. The leakage of Cu(II) from injectable microcryogels was investigated using a graphite furnace atomic absorption spectrometer (AAS), specifically the Analyst 800 model by Perkin-Elmer in Shel- ton, CT. The outcomes are obtained n = 3 considering of environment and drug handling behavior, and mean values are denoted. Imprinted microcryogels were investigated for different loaded DRV concentrations. The concentration of the DRV was determined at 280 nm. Shimadzu, Model 1601, Tokyo, Japan, UV/Vis spectrophotometer is used for the entire experiment. Also, to show the effect of pH on the cumulative release of DRV, the effect of pH was inspected at different pH values. In order to see the effect of cross- linker and monomer ratio at different ratios, the cryogels with the mole ratios of HEMA and MBAAm 4, 6, 8 were synthesized. As the cross-linker ratio increased, the release rate decreased. In order to investigate the effect of release at different pHs, drug release from DRV loaded microcryogels at 4 pHs (6.0, 7.0, 7.4, and 8.0) was investigated. (1)Swelling % = ( Wswelled − Wdry ) ∕Wdry) 4011Chemical Papers (2024) 78:4007–4018 Cytotoxicity studies Biological evaluation of medical devices' standards of ISO- 10993–5 was used to investigate the cytotoxic effect and 3-(4,5-dimethyl/thiazol-2-yl) 2,5-diphenyltetra zolium bro- mide thiazolyl blue (MTT) test was used and fibroblast cell line (L929) was used. The culture medium of the cells was established with 10% fetal bovine serum and 10% L-glu- tamine in a humidified atmosphere containing 95% air and 5% CO2 at 37 °C containing DMEM and lasted for 3 days. Ultraviolet light was used for the sterilization of cryogels and they were incubated for 72 h at 37 °C L929 cells have densities of 1 × 103 and these cells were cultured in 96 wells with a volume of 200 µL and incubated overnight in a humidified medium with 5% CO. The cell culture medium was then replaced with the extraction medium and incubated again at 37 °C for 24 h. Cultured cells were treated with 100 µL/well of MTT solution for 4 h. The plates were then incubated at room temperature for 30 min in a dark place. Finally, the wavelength of 540 nm was read by an automated enzyme-linked immunosorbent assay (ELISA). Results and discussion In this study, an antiretroviral drug, DRV-imprinted micro- cryogels, and its release kinetics were investigated. DRV- imprinted pHEMA-based microcryogels were synthesized with varying porosity by using polymer precursors with dif- ferent properties. In addition, the effect of loading amount was observed by adding different amounts of DRV, while preparing the microcryogels. Then, the release kinetics and cytotoxicity of the prepared microcryogels were investi- gated. Experimental methods were carried out in three basic steps: preparation of DRV-imprinted and non-imprinted pHEMA-based microcryogels (MIP and NIP microcryo- gels), characterization of DRV-imprinted and non-imprinted pHEMA-based microcryogels, and investigation of DRV drug release under in vitro conditions. Characterization of microcryogels In drug removed MIP and NIP microcryogel spectra, 3348 and 3407  cm−1 O–H stretching peaks are observed. Besides, peaks at 2950 and 2934  cm−1, respectively, were seen as C–H stretching for aliphatic alkyl. C=O stretching made its presence known with the peaks at 1714 and 1725  cm−1, and N–H stretching stands outs with the peaks at 1658 and 1661  cm−1 from MAH content of the cryogels. The peaks at 1452–1450  cm−1 and 1384–1392  cm−1 are indicative of C–H stretching. In addition, 1240–1252  cm−1 peaks and 1148–1151  cm−1 peaks are stands for C–O stretching also from MAH content. The peaks at 1071 and 1081  cm−1 are C–O stretching from primary alcohol presence. All these peaks have not only revealed their HEMA content but also indicated that MAH thoroughly entered the polymeric struc- ture. In HEMA specific case, 3348  cm−1 O–H stretching, 2947  cm−1 alkane C–H stretching, and 1723  cm−1 peaks are C=O stretching, and 1071  cm−1 is presence of primary alcohol (Fig. 2). Three different cross-linker ratios were synthesized to examine the effects of pore structure and surface area of microcryogels on drug release behavior. The surface mor- phology of the microcryogels prepared within the scope of this study was examined by scanning electron microscopy and structural morphology by microscope. Optical images and SEM images of microcryogels are given in Fig. 3 and 4. In Fig. 3, the optical image of microcryogels is shared and it is shown that they can pass through a standard injector without any blockage. In Fig. 4 SEM images, there is a uni- form and homogeneous distribution within the microcryogel polymer structure. The SEM figures illustrate the extensive interconnected pores within the microcryogels. According to BET measurements, the specific surface area for MIP I, MIP II, MIP III, MIP IV, NIP, and pHEMA microcryo- gels was determined to be 14.1, 14.9, 15.3, 16.1, 12.8, and 12.1  m2/g, respectively. Figure 4 demonstrates the highly porous nature of the structure of the microcryogels, aligning with the surface area findings. Due to these macropores, it has been determined that the channels are interconnected in the cryogel structure. Macropores facilitate the diffusion of water from the microcryogel structure into the polymeric material and to the external environment during the swelling and shrinking processes. This reversible swelling-shrinkage behavior of macropo- rous microcryogels is considered an important advantage in biomaterials and biotechnology applications. The effect of drug loading rate on swelling behavior and surface area was investigated for microcryogels synthesized at −14 °C. The surface area and swelling behavior of microcryogels are compiled in Table 1 at different loading rates. It can also be observed that both the swelling ratio and the surface area increase significantly with an increase in the DRV loading amount. Increasing the DRV load- ing amount from 0.5 to 1.75 mg increased the swelling rate from 8.17 to 9.88%. An almost 1.2-fold increase in swelling rate has been reported to be consistent with an increase in surface area values. The surface area value of 0.5 mg DRV loaded pHEMA microcryogel was 11.7  m2/g, while this value was reported as 18.5  m2/g for 1.75 mg DRV loaded. The increase in surface area is an important parameter in DRV release studies, controlling its rate and also in releasing the amount of DRV. The increase in the structural pores of microcryogels causes this behavior. As a result of this increase, the amount of water entering the structure of microcryogels increases. In addition, 1.0 mg 4012 Chemical Papers (2024) 78:4007–4018 of MIP and NIP microcryogels were kept separately at pH 4.5, pH 7.4, and pH 8.5 for 24 h in order to control the leakage of Cu(II) ions from the structure of MIP and NIP microcryogels, and then the metal ions concentra- tion of the solution taken from the environment was read. Copper(II) ion readings were determined through atomic absorption spectroscopy (AAS). The analytical reports yielded no detection of Cu(II) ions in any of the examined pH solutions. Based on these findings, it can be inferred that the pre-complex exhibits stability. Therefore, it has been explained that in drug release applications, Cu(II) ions are not released from the structure of microcryogels and cannot cause any toxic effects. Investigation of Darunavir release behavior from MIP microcryogels Investigation of the effect of Cu(II) ion in different ratios on the release rate In the preparation of microcryogels, the effect of the increase in Cu(II) on the release rate of DRV was inves- tigated by preparing 3 different mole ratios of MAH- Cu(II) pre-complex separately and selecting 3 different DRV-imprinted microcryogels. At this stage, the mole ratio of MAH functional monomer is kept constant and the amount of Cu(II) ions is 1MAH-1Cu(II)- DRV, Fig. 2 FTIR spectrum of DRV-imprinted (MIP), non-imprinted (NIP) microcryogels and pHEMA Fig. 3 Optical images of micro- cryogels 4013Chemical Papers (2024) 78:4007–4018 1MAH-2Cu(II)-DRV, and 1MAH-3Cu(II)-DRV, 1:1, 1:2, and changed to 1:3. In addition, while the amount of DRV loaded was 1.5 mg/mL, the release was at 37 °C by keep- ing the pH of the release at 7.4. The graph of the release rate is given in Fig. 5. Due to the results obtained, the mole ratio of MAH-Cu(II)-DRV to be used throughout the research was chosen as 1:1:1, since the release rate of DRV is further controlled at a mole ratio of 1:1:1 and 62%. Fig. 4 SEM images of micro- cryogels Table 1 Effect of swelling behavior and surface area of microcryogels loaded with different amounts of DRV Microcryogels Amount of loaded drug (mg DRV/ mg microcryogel) Swelling rate (%), mg H2O/mg microcryogel Surface area (m2/g) MIP I 0.5 8.17 14.1 MIP II 1 8.65 14.9 MIP III 1.5 9.06 15.3 MIP IV 1.75 9.88 16.1 NIP – 8.01 12.8 pHEMA – 7.65 12.1 4014 Chemical Papers (2024) 78:4007–4018 Investigation of the effect of cross‑linker at different rates on DRV release rate To determine the effect of cross-linker and monomer ratio at different ratios, 3 different microcrystals were prepared and the release test was performed with these microcryogels. The effect of the cross-linker is shown in Fig. 6 with a molar ratio of 4, 6, and 8, respectively. Since the cross-linker has a significant role in determining the polymer structure and pore size, its effect on the drug release rate should also be observed. DRV was released from microcryogels at a fixed amount of drug (1.5 mg/ mL). Figure 6 indicates that the controlled release of DRV is tied to the concentrations of MBAAm and the cross- linker, exhibiting an inverse correlation. A high density of the cross-linker causes the microcryogel to become more rigid due to shrinkage and reduced voids in the micro- cryogel network. The increased amount of the cross-linker leads to the harder structure of the microcryogels due to the narrowing and reduction of the voids in the polymer network. This may have resulted in increased stiffness of the microcryogel in the unfrozen areas as the number of cross-linker increases. At this stage, the molar ratio of MAH-Cu(II)-DRV was used as 1:1:1. The drug amount was chosen 1.5 mg/mL, release pH: 7.4, and release tem- perature 37 °C. Fig. 5 Effect of Cu(II) in at different ratios on the release rate, release medium pH: 7.4; temperature: 37 °C 0 20 40 60 80 100 0 10 20 30 40 50 D R V R el ea se (% ) Time (hour) 1MAH-1Cu(II)-1DRV 1MAH-2Cu(II)-1DRV 1MAH-3Cu(II)-1DRV Fig. 6 Effect of different cross- linker and monomer ratios on the release rate, release medium pH: 7.4; temperature: 37 °C 0 20 40 60 80 100 0 10 20 30 40 50 D RV R el ea se (% ) Time (hour) n:4 n:6 n:8 4015Chemical Papers (2024) 78:4007–4018 Investigation of the effect of drug loading amount on drug release rate Darunavir imprinted microcryogels were prepared by load- ing 0.5–1.75 mg/mL DRV drug into MIP microcryogels. DRV release rates from microcryogels loaded with different amounts of drug (0.5, 1.0, 1.5, and 1.75 mg DRV/mg micro- cryogel) are shown in Fig. 7 and state that the DRV cumula- tive release increases with the increase in the amount of drug in DRV-imprinted microcryogels. Drug release rate of 25% was obtained when DRV loading amount was 0.5 mg DRV/ mg microcryogel. This ratio was 40, 70, and 85% for 1.0, 1.5, and 1.75 mg DRV loaded microcryogels, respectively. Almost all of the drugs in the microcryogel structure were released from the structure in microcryogels loaded with 1.75 mg. The concentration difference of the drug releases DRV from the microcryogel structure is the driving force of the mass transfer. Therefore, the driving force is propor- tional to the concentration of the drug. After the initial rapid release, the drug reached a plateau, maintaining a consistent level across all drug release loadings. The figure indicates that the release duration of DRV from microcryogels has the potential to be extended up to 40 h. Effect of pH on Darunavir release rate To examine the effect of pH on release rate, different media pHs of 6.0, 7.0, 7.4, and 8.0 are investigated in Fig. 8. In compliance with the obtained results, the maximum and controlled and steady drug release is obtained at pH 7.4. In cases where Cu(II) ions are used in metal chelate systems, it is expected that DRV release will be lower at acidic pH, since the acidic pH of the environment increases the stability of the metal chelate. In the basic environment, the release occurred faster because the interactions between metal ions and the drug were weaker. Acidic pH values are not pre- ferred considering the possibility of damaging the structure of drugs. Therefore, the physiological pH of 7.4 is preferred for release studies. To understand the effect of the pH on the release rate, different pHs (pH: 6.0–8.0) media are prepared. The release test was carried out with MIP microcryogels (selecting n:4 and the pre-complex ratio 1:1:1 for MAH- Cu(II)-DRV). Release experiments were performed at 37 °C when the DRV imprinting amount was 1.5 mg/mL. Analysis of release kinetics The Korsmeyer–Peppas model was chosen to analyze the in vitro drug release kinetics and mechanism. In the Korsmeyer–Peppas model, the value of n describes different release mechanisms from cylindrical-shaped matri- ces and k is the release rate constant. Mt is the DRV release amount at time t, and the M∞ is the release at the equilib- rium point. To sum up, Mt/M∞ is the drug release fraction. In Table 2 R2, correlation coefficient constants and DRV conduction exponent data are presented. According to the n value of Eq. 2, n = 0.5 implies simple Fickian diffusion mechanism. However, calculated n values for MIP II, MIP III, and MIP IV are below 0.5 implying that drug release mechanism for these cryogels is complex Fick- ian diffusion. The main reason for complex Fickian diffusion is releasing drug through the swollen hydrogel and water- filled pores and that is also the case for this study. Although (2)M t ∕M∞ = kt n Fig. 7 Effect of DRV loading amount on the drug release rate, release medium pH: 7.4; temperature: 37 °C 0 20 40 60 80 100 0 10 20 30 40 50 D RV R el ea se (% ) Time (hour) 1.75 mg/mL 1.5 mg/mL 1.0 mg/mL 0.5 mg/mL 4016 Chemical Papers (2024) 78:4007–4018 swelling effect results in complex Fickian diffusion, MIP I hydrogel shows that more swelling changes the diffusion mechanism from complex Fickian to non-Fickian. When smaller amount of drug is loaded (MIP I case), water pen- etration to cryogel is increased because of non-loaded pores. In that case, the calculated n value for MIP I is above 0.5 indicating that diffusion mechanism becomes non-Fickian (Korsmeyer et al. 1983; Bacaita et al. 2014). Cytotoxicity studies The mouse fibroblast cell line L929 was also used to meas- ure the cytotoxicity of MIP, NIP, and pHEMA microcryo- gels. Cytotoxicity results were determined as 95.02 ± 0.13, 96.11 ± 0.11, and 97.02 ± 0.10 for MIP, NIP, and pHEMA samples after 24 h. These rates indicate that MIP and NIP microcryogels do not have a toxic effect on the L929 cell line as a drug carrier material. The toxicity of pHEMA as a based material is less than MIP and NIP cryogels. MAH-Cu content cryogel content used as functional monomer may explains the slight difference of the cell viability between MIP-NIP cryogels and pHEMA. The detailed results are shown in Fig. 9. Conclusion This study is a successful combination of molecular imprinting and cryogel applications and is presented as an alternative and effective method for controlled drug release. Controlled release of imprinted DRV in pHEMA microcryogels was investigated at different parameters such as pH, drug concentration, and various cross-linker and monomer ratios. The results showed that the con- trolled release of DRV from microcryogels was promising in terms of and adjustability yet steady kinetics. Addition- ally, biocompatibility of microcryogels has been proved with cytotoxicity tests. In conclusion, this study not only marks a successful integration of molecular imprinting and cryogel applications for controlled drug release but also presents a promising path for future advancements in the field. Fig. 8 Effect of pH on DRV release rate, temperature: 37 °C 0 20 40 60 80 100 0 10 20 30 40 50 D RV R el ea se (% ) Time (hour) pH: 6.0 pH: 7.0 pH: 7.4 pH: 8.0 Table 2 Compilation of release kinetic data according to the Korsmeyer–Peppas model Microcryogels Amount of loaded drug (mg DRV/mg microcryo- gel) MAH-Cu(II)- DRV mole ratio Cross-linker/ monomer ratio (n) n k R2 MIP I 0.5 1:1:1 4 0.53 0.09 0.98 MIP II 1.0 1:1:1 4 0.41 0.16 0.98 MIP III 1.5 1:1:1 4 0.40 0.25 0.98 MIP IV 1.75 1:1:1 4 0.35 0.39 0.98 4017Chemical Papers (2024) 78:4007–4018 Declarations Conflict of interest All authors declare that there are no conflicts of interest. References Aydin D, Arslan M, Sanyal A, Sanyal R (2017) Hooked on cryogels: a carbamate linker based depot for slow drug release. 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