Nanogels with high loading of anesthetic nanocrystals for extended duration of sciatic nerve block

Nanogels with High Loading of Anesthetic Nanocrystals for Extended Duration of Sciatic Nerve Block Teresa Alejo,*Laura Uson, Guillermo Landa, Martin Prieto, Cristina Yus Argón, Sara Garcia-Salinas, Ricardo de Miguel, Ana Rodríguez-Largo, Silvia Irusta, Victor Sebastian, Gracia Mendoza,* and Manuel Arruebo Cite This: ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 Read Online ACCESS Metrics & More Article Recommendations * sıSupporting Information ABSTRACT: The development of thermoresponsive nanogels loaded with nanocrystals of the local anesthetic bupivacaine nanocrystals (BNCs) for prolonged peripheral nerve pain relief is reported here. BNCs were prepared using the antisolvent precipitation method from the hydrophobic form of bupivacaine (bupivacaine free base). The as-prepared BNCs were used standalone or encapsulated in temperature-responsive poly(ethylene glycol) methyl ether methacrylate (OEGMA)-based nanogels, resulting in bupivacaine NC-loaded nanogels (BNC-nanogels) of monodisperse size. The synthesis protocol has rendered high drug loadings (i.e., 93.8 ±1.5 and 84.8 ±1.2 wt % for the NC and BNCnanogels, respectively) and fast drug dissolution kinetics in the resulting composite material. In vivo tests demonstrated the efficacy of the formulation along with an extended duration of sciatic nerve block in murine models of more than 8 h with a formulation containing only 2 mg of the local anesthetic thanks to the thermoresponsive character of the polymer, which, at body temperature, becomes hydrophobic and acts as a diffusion barrier for the encapsulated drug nanocrystals. The hydrophobicity of the encapsulated bupivacaine free base probably facilitates its pass through cell membranes and also binds strongly to their hydrophobic lipid bilayer, thereby protecting molecules from diffusion to extracellular media and to the bloodstream, reducing their clearance. When using BNC-nanogels, the duration of the anesthetic blockage lasted twice as long as compared to the effect of just BNCs or a conventional bupivacaine hydrochloride solution both containing equivalent amounts of the free drug. Results of the in vivo tests showed enough sensory nerve block to potentially relieve pain, but still having mobility in the limb, which enables motor function when required. The BNC-nanogels presented minimal toxicity in the in vivo study due to their sustained drug release and excellent biocompatibility. The encapsulation of nano-sized crystals of bupivacaine provides a prolonged regional anesthesia with reduced toxicity, which could be advantageous in the management of chronic pain. KEYWORDS: bupivacaine nanocrystals, local anesthesia, nerve blockade, drug delivery, thermoresponsive nanogels ■INTRODUCTION Pain treatment is a clinical challenge owing to the short duration of anesthetic effects, associated with the low molecular weight of the drugs, and the potential systemic toxicity provided by current existing treatments. 1 At present, clinical local or regional anesthetic effects rarely last beyond 12 h unless in case of using continuous catheter infusions. 2,3 Most common prescriptions are based on antipyretic analgesics and opioids; nevertheless, these medications present severe adverse side effects, such as dizziness, nausea, vomiting, constipation, physical dependence, and respiratory complications. 4,5 The development of an effective injectable local anesthetic with prolonged duration of action would enhance the quality of life of patients affected by chronic or postoperative pain. 6 Conventional free drugs present several disadvantages that impair their optimal performance, such as short half-life in the organism, high dosages administered, poor selectivity, and associated side effects in healthy tissues. The incorporation of drugs into nanoparticles can improve the therapeutic characteristics of the formulations and outweigh some of those drawbacks. Drug nanoencapsulation has been demonstrated to reduce its systemic toxicity and opens up the possibility of adding targeting characteristics that enhance the accumulation of the nanomedicine at diseased sites. 7 However, for an Received: January 14, 2021 Accepted: March 25, 2021 Published: April 6, 2021 Research Articlewww.acsami.org © 2021 American Chemical Society 17220 https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 Downloaded via CSIC on April 1, 2022 at 08:52:41 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. efficient treatment, the amount of drug loaded into the carrier nanomaterial must be high enough. Usually, nanomedicines have shown low drug-loading contents (in many cases less than 10 wt %). 8−12 Therefore, efforts must be made to develop nanomedicines in which the ratio of payload to nanomaterial could be increased. Nanomedicines with high drug loadings (DLs) can deliver large amounts of drug per nanomaterial unit. Also, the reduction of the nanoparticulate carrier in the total nanomedicine content can decrease the overall nanomaterial fabrication cost and also reduce the potential side effects associated with the carriers themselves. 13 Drug loading mechanisms driven by supramolecular interactions such as electrostatic and physical adsorption often outcome low drugloading efficiencies; meanwhile, high drug loadings can be obtained in the case of crystallization and when using covalent and coordinative bonds. 14−17 However, chemical modification of the transported drug should be avoided to preserve its therapeutic function. Nanocrystals (NCs) have been used during the past 20 years to improve the bioavailability and dissolution rate of poorly Figure 1. (a) Scheme of the global synthesis of BNC-nanogels. The first stage is the preparation of BNCs using antisolvent precipitation method. Second is the synthesis of thermoresponsive nanogels by in situ free-radical copolymerization of MEO2MA and OEGMA500 monomers in the presence of as-prepared BNCs. (b) Representative TEM image of thermoresponsive nanogels. (c) Size distribution histogram with Gaussian-fitting curve (solid line) for nanogels obtained from TEM images (N= 80). The average size was 64.7 ±8.6 nm. (d) Size distribution dynamic light scattering (DLS) plots obtained for nanogels at different temperatures. (e) Variations in the hydrodynamic sizes and volume change factor of nanogels as a function of temperature measured using DLS. Data are mean ±standard deviation (SD) (N= 5). (f) TEM image presenting BNCs size and morphology; inset: size distribution histogram obtained from TEM images (N= 80). The average size was 21.9 ±7.2 nm. (g) X-ray diffraction (XRD) diffractograms of bupivacaine, BNCs, and HPMC. ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17221 water-soluble hydrophobic drugs in several formulations intended for oral delivery. 18,19 Nanocrystallization is recommended for the enteral delivery of drugs belonging to the Class II Biopharmaceutics Classification System (i.e., high permeability and low solubility). In this way, by changing the size to the nanosize range, the specific surface area is increased, giving an enhancement in the rate of dissolution in physiological media. 20 In fact, several Food and Drug Administration (FDA) approved nanocrystal-based drugs are already available on the market. 18 Those commercially available products are usually prepared by top−down methods, including media milling and high-pressure homogenization. 21,22 The number of FDAapproved products based on NCs prepared by bottom-up methods is very limited, although they have been demonstrated to potentially produce narrow NCs using low-cost scalable procedures. For instance, Liu et al. 23 prepared NCs of two antitumoral drugs, paclitaxel and camptothecin, using Pluronic 127 as a stabilizer to increase the circulation lifetimes of both drugs. They achieved lower toxicity than the equivalent dose of the free drugs, and an excellent antitumor activity, besides, an easy scale-up manufacturing method was proposed. Antisolvent precipitation methods have been used to obtain NCs around 200 nm in size having different stabilizers, usually methylcellulose derivatives, 24,25 poly(vinyl alcohol) (PVA), 26 using various solvent/antisolvent combinations and also, in some cases, even without the use of any stabilizer. 27 Stable NCs of mean particle sizes in the range of 10−20 nm were obtained using poly(ethylene glycol) (PEG300) as solvent for the dissolution of the drug (carbamazepine), and an aqueous solution of hydroxypropyl methylcellulose (HPMC) as antisolvent. 28 The presence of the cellulose derivative inhibits crystal growth, and was found to decrease the particle size by its adsorption on the surface of hydrophobic drugs limiting the access for the dissolved drug in the medium to the growing crystallization nuclei. The high surface activity of methoxy and hydroxypropyl groups present in cellulose derivatives allows the interaction with drug molecules via hydrogen bonding, which inhibits crystal growth rendering crystals of a few nanometers in diameter, being nucleation promoted and crystal growth arrested. NCs have also been loaded within polymeric particles to promote their sustained drug release. For instance, Wang et al. 29 developed poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) loaded with breviscapine (a naturally occurring flavonoid) NCs, using a water-soluble polymer template method, having an improved drug loading for a prolonged drug delivery representing a promising approach for long-term delivery of therapeutic doses. Several nanomaterials have been proposed as drug delivery systems to obtain long-term analgesia in the management of chronic pain. 30 A sustained-release system with long-term action is desired to satisfy the patients’requirements, as well as to maintain constant therapeutic levels. Current available local anesthetics approved for single-injection with extended duration of action are, for instance, liposomal formulations of bupivacaine (Exparel) and morphine (DepoDur). Furthermore, on-demand triggerable nanoparticulated systems for achieving pulsatile anesthetic release profiles have also been reported. For instance, phototriggered local anesthesia was achieved using liposomes attached to gold nanorods as plasmonic nanoparticles that generate heat upon light stimulation to trigger the release of a local anesthetic drug (i.e., tetrodotoxin) on-demand. 31 Herein, to achieve long-term local anesthesia, bupivacaine nanocrystals (BNCs) were prepared and encapsulated within biocompatible poly(ethylene glycol) methyl ether methacrylate (OEGMA)-based nanogels. To the best of our knowledge, this is the first time that BNCs are prepared and tested in vivo for pain management applications and benchmarked against the clinically used bupivacaine hydrochloride solution. One of the objectives of this study was to synthesize thermoresponsive biocompatible nanogels with high drug loadings. The ability of the nanogels loaded with bupivacaine nanocrystals (BNCnanogels) to provide in vivo sciatic nerve blockade was successfully evaluated. Moreover, the potential in vitro and in vivo toxicity of the materials was assessed in this study. ■RESULTS AND DISCUSSION Characterization of Nanogels. The preparation of thermoresponsive nanogels was performed in one-pot synthesis by in situ free-radical copolymerization of MEO2MA and OEGMA500 monomers, according to the procedure described in the Experimental Section, 32 and as it is depicted in Figure 1a. Transmission electron microscopy (TEM) images of empty (i.e., drug-free) nanogels in Figure 1b show particles with spherical-like morphology of uniform size. The size distribution histogram with Gaussian-fitting curve (solid line) for nanogels shown in Figure 1c exhibits a narrow size distribution. The average size of the empty nanogels measured from TEM images (N= 80) was 64.7 ±8.6 nm. The ζ potential of the nanogels dispersed in Milli-Q water was negative, with a value of −6.7 ±0.9 mV. The negative surface charge is attributed to the sulfate groups present in the anionic surfactant used to stabilize the nanoparticles together with the steric hindrance of the OEGMA groups. Indeed, the nanogels obtained showed great stability, as they remained dispersed in solution during weeks without sedimentation and showing the same hydrodynamic size and ζpotential after at least 3 months of storage (results not shown). Also, their stability in water is the reason for the use of ultracentrifugation at 25 000 rpm to precipitate them and recover the nanogels from water. The thermoresponsive properties of nanogels were evaluated using DLS. To accomplish this, the change in the hydrodynamic average size of nanogels dispersed in Milli-Q water was determined at different temperatures from 22 to 65 °C. Before each DLS measurement, the temperature was equilibrated in the sample holder at least for 5 min. The hydrodynamic size distributions shown in Figure 1d are highly monodisperse, with polydispersity index (PDI) values between 0.001 and 0.09. Temperature-induced shrinking of nanogels was observed in the plot. Hydrodynamic nanoparticle sizes are consistent with TEM results, as the larger size obtained by DLS is explained by the effective swelling of the nanogels in aqueous dispersions. Conversely, the smaller size obtained by TEM is the one of the dry dehydrated shrunken nanogels under the high-vacuum conditions used. The thermoresponsive behavior of the obtained nanogels is presented in Figure 1e with the plot of hydrodynamic size versus temperature, where the reduction in the size of nanogels with the increase in temperature is due to the collapse of the nanogels above their volume phase transition (VPT). The inflection point of the hydrodynamic size versus temperature curve is defined as the volume phase transition temperature (VPTT) of the nanogels. The nanogels with VPTT above the physiological temperature (37 °C) are suitable as drug carriers. The VPTT obtained was 38.3 °C, which is in accordance with ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17222 our previous studies on lower critical solution temperature (LCST) of the equivalent OEGMA-based copolymers. 33 The produced nanogels were able to reduce their size from 107.9 nm at 25 °C (swollen) to 72.1 nm at 60 °C (shrunken), giving a volume change factor of 3.3. Therefore, they undergo 70% reduction in their volume and hydrophilic/hydrophobic character, which allows them to be used as drug vehicles to release their cargo upon temperature changes in a reversible manner. Furthermore, rheological analysis was performed to study the phase transition of the nanogels (Figure S1). VPTT can be determined through tracking the evolution of storage modulus (G′) and loss modulus (G″) in dynamic temperature sweep tests. Results in Figure S1 show that below the transition temperature G′and G″remained constant with temperature; however, when the phase transition was reached, the enhancement of the system elasticity resulted in a sharp increase in G′and G″moduli. The critical temperature at which G′and G″rapidly increase (40.5 °C) is considered the VPTT. When the temperature increases until reaching the VPTT, intermolecular and intramolecular attractive interactions between polymer chains increase and dehydration occurs, which causes an increase in the rigidity of nanogels and therefore an abrupt increase in G′and G″. 34 The slight difference in the VPTT obtained by DLS and by rheological analysis can be ascribed to the difference in the analytical technique used, since in DLS, we determined the volume change of nanogels as colloidal suspensions, and in rheological tests, we analyzed the changes in their viscoelastic properties. Nevertheless, both methods displayed consistent results, giving a close value for VPTT. Characterization of BNC-Nanogels. With the aim to obtain high drug loading contents, the local anesthetic bupivacaine was nanocrystallized. The NCs were prepared from the hydrophobic form of bupivacaine (bupivacaine free base) using the antisolvent precipitation technique, before being introduced in the precursor solution for the synthesis of nanogels. As far as we know, this is the first time that BNCs are reported. TEM studies were performed to examine the size distribution and morphology of the obtained BNCs. TEM images showed spherical NCs of 21.9 ±7.2 nm in size with a Figure 2. Electron microscopy analysis of BNC-nanogels: (a) representative TEM images of BNC-nanogels. (b) Size distribution histogram obtained from TEM images with a number of elements N= 80. The average size was 148 ±35 nm. (c) High-resolution TEM image showing in detail a nanogel embedding BNC. (d) Photograph of BNC-nanogels dispersed in aqueous solution. (e) XRD patterns recorded for BNCs, nanogels loaded with BNCs and empty nanogels. (f) TGA (black) and derivative of TGA (blue) plots for BNC-nanogels. (g) Bupivacaine release profiles from BNCs (nonencapsulated) and BNC-nanogels at 37 °C. Data are mean ±SD (N= 3). ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17223 narrow size distribution (Figure 1f). The XRD technique was used to characterize the BNCs crystallinity and the size of their elementary crystallites (i.e., unit cell dimension). The most representative XRD diffraction planes of bupivacaine reference (Figure 1g) were located at 10, 16.2, 17.8, 20, 21.5, 23.8, and 25.5°. The spectrum of the obtained BNCs matched most of the reference diffraction planes, indicating that the BNC structure remains in a well-crystallized state. The XRD spectra of HPMC used as a stabilizer agent and empty nanogels showed the characteristic plots of amorphous compounds. The mean size of the BNCs elementary crystallites can be determined using Scherrer’s equation. 35 Analyzing the five most intense peaks of the spectra, the size of BNCs obtained using the Scherrer constant for spherical crystallites (K= 0.94) using the Voigt fitting model was 35.93 ±1.1 nm. This value is in good agreement with the microscopy characterization. The drug loading obtained by GC-MS for BNCs was 93.8 ±1.5 wt %. BNCs are therefore mainly consisting of drug crystals having a small fraction of HPMC. To obtain BNC-nanogels of high drug content, the BNCs were introduced during the synthesis of the nanogels, with the purpose of growing the nanogels on the surface of the crystals. The encapsulation of NCs could be mainly driven by hydrophobic interactions of highly methylated glucose zones in HPMC 36 with the main polymer backbone having hydrophobic character, keeping the drug in the core of the nanoparticles. The overall scheme representing the synthesis of BNC-nanogels is depicted in Figure 1a. Figure 2a,b shows TEM images and size distribution histogram of BNC-nanogels, respectively. It can be seen that nanogels presented a uniform size with a high electronic density in the core due to the BNCs embedded in their interior (Figure 2a,c). The size of the BNC-nanogels was 148 ±35 nm. This size is twice the one of empty nanogels presented in Figure 1b. The considerably larger size of the BNC-nanogels indicates the encapsulation of the drug crystals inside. Photographs of the obtained BNC-nanogels dispersed in aqueous solution are shown in Figure 2d. The XRD characterization of BNC-nanogels in Figure 2e shows the characteristic bupivacaine diffraction planes, which demonstrates that nanocrystals were present in the nanogels. The ζ potential of the BNC-nanogels remains in the same range as the one for empty nanogels, with a value of −6.91 ±0.38 mV. BNC-nanogels were analyzed by thermogravimetric analysis (TGA) in the temperature range of 30−800 °C. Figure 2f shows the weight loss of the nanogels embedding BNCs as a function of temperature. The thermal decomposition of the BNC-nanogels occurred between 150 and 400 °C. At 400 °C, the weight loss corresponds to the total initial mass. Bupivacaine hydrochloride total thermal decomposition takes place at 266 °C(seeFigure S2), and the polymer P(MEO2MA-co-OEGMA500) decomposition occurs between 250 and 400 °C. 37 The derivative of the weight loss (Figure 2f) presented two defined peaks centered at 260 °C, attributed to the bupivacaine thermal decomposition, and at 340 °C, compatible with the polymer loss (Figure S2). Therefore, the polymer content in the final formulation and drug content Figure 3. Cell viability of BNC-nanogels, BNCs, empty drug-free nanogels, and bupivacaine hydrochloride in the four cell lines assayed after 24 h. The red line depicts the threshold of 70% of cell viability in accordance with ISO 10993-5. Percentages are displayed as mean ±SD (N= 5). ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17224 were estimated to be 13 wt % and 87 wt %, respectively. The partial overlap of the pure polymer and drug curves does not allow us to precisely determine the mass of each component by this technique; however, the estimated value was consistent with the DL% obtained using gas chromatography−mass spectrometry (GC-MS). The quantitative determination of the drug incorporated into nanogels and its posterior in vitro release were performed by GC-MS. The drug loading of BNC-nanogels determined by GC-MS was calculated as 84.8 ±1.2 wt %. To the best of our knowledge, this is one of the highest bupivacaine loadings reported in polymeric nanoparticles, even superior to the results obtained by Curley et al. 2 who obtained PLGA microspheres loaded with 75 wt % bupivacaine free base using oil-in-water (o/w) emulsification and solvent evaporation method. The drug release profiles of BNC-nanogels and BNCs are shown in Figure 2g. The dispersions were prepared to have the same drug amount in both samples according to the drug loading (0.196 mg for BNC-nanogels and 0.180 mg for BNCs in 1 mL aqueous solution) and to work below the drug saturation concentration of the bupivacaine free base form (0.4 mg/mL) as working under sink conditions. 38 Results showed a fast release with an onset reached in less than 30 min when the drug concentration was below the saturation concentration. As it is shown in Figure 2g, the release of BNCs and BNC-nanogels is quite similar and the dissolution of the drug occurs in the first hours under conditions below the drug saturation concentration. The dissolution rate of nanocrystals composed of the bupivacaine free base will be limited by the low aqueous solubility of this nonionized form (0.4 mg/mL), in contrast to bupivacaine hydrochloride salt having a solubility 10-fold higher (20−40 mg/mL). 38 To analyze the kinetics of drug release from BNCs and BNCnanogels, release results were fitted to different mathematical models: zero order, first order, Higuchi, Korsmeyer−Peppas and Hixson−Crowell. The best model was selected according to the correlation coefficient (R2) determined from the linear regression fit for each model. The obtained kinetic data for the drug release together with the correlation coefficients (R2) are listed in Table S1. According to the regression results, the release profile of BNC-nanogels (R2= 0.998) and BNCs (R2= 0.999) followed the Korsmeyer−Peppas model with kinetic constant KBNC‑nanogels = 0.884 h−nand KBNCs = 0.963 h−n. The value of the kinetic constant decreased when BNCs were encapsulated in the nanogels due to the increased viscosity and governance of polymeric chain entanglement. 39 The parameters obtained for the release exponent (nBNC‑nanogels = 0.041 and nBNCs = 0.021) less than 0.5 indicate that the drug release was governed by Fickian diffusion as reported by other authors for thermoresponsive polymeric nanoparticles. 40 The initial rapid drug release has been attributed to the fast dissolution rate of small NCs 41 and the presence of drug molecules that are located close to the external surface, but embedded in the polymeric matrix. 42 In Vitro Biological Assays. The in vitro cytotoxic effects of the synthesized materials were studied in four different cell lines: human dermal fibroblasts, macrophages, mouse mesenchymal stem cells (mMSCs), and U251MG. After treatment for 24 h, the effects on cell metabolism, apoptosis, and cell cycle were assessed. Moreover, the endotoxin levels of BNCs and BNC-nanogels were evaluated to ensure their safety for their in vivo application. The treatment of the cell lines described above for 24 h with BNC-nanogels, BNCs, empty drug-free nanogels, and bupivacaine hydrochloride solution produced the effects depicted in Figure 3. The assays were performed at BNCnanogel concentrations ranging from 0.01 to 0.5 mg/mL and at the equivalent drug concentrations, for BNCs and free bupivacaine hydrochloride, or the equivalent polymer concentration in the case of empty nanogels. Empty thermoresponsive nanogels did not reveal cytotoxic effects as cell viability was found in all concentrations and cell lines tested above 92% in compliance with ISO 10993-5, 43 which describes that a reduction in cell viability higher than 30% compared to the control sample is considered as cytotoxic. These results are in accordance with previous results of our group in which the thermoresponsive polymer P(MEO2MA-co-OEGMA500) synthesized by photopolymerization exerted cell viability percentages higher than 92% at concentrations up to 0.4 mg/mL. 33 On the other hand, BNCs and bupivacaine hydrochloride involved a decrease in cell viability (∼50%) at the highest concentration assayed (0.5 mg/mL) except for THP1-derived macrophages, which maintained cell viability in the same range as the control sample. Finally, BNC-nanogels also showed some cytotoxic effects in U251MG and mMSCs at the highest concentration studied displaying viabilities around 59 and 50%, respectively. However, percentages higher than 70% were recorded in fibroblast and macrophage samples at all of the concentrations assayed. It should be noted that at the highest concentration assayed, BNC-nanogels displayed higher viability percentages than BNCs pointing to the potential protective effect of the nanogel by including inside its structure the BNCs, except for macrophages whose percentages were in the range of the control sample (100%). With these results, the subcytotoxic dose for further experiments for BNC-nanogels, BNCs, and bupivacaine hydrochloride was considered 0.1 mg/ mL, whereas for the empty nanogels, 0.5 mg/mL was selected. Previous works in our group have revealed similar results regarding the treatment of these cell lines with bupivacaine loaded in hollow gold nanoparticles (HGNPs) functionalized with the thermoresponsive polymer disulfide-P(MEO2MA-coOEGMA500), 37 bupivacaine loaded in a cleavable nanocomposite composed of CuS nanoparticles and POEGMA (CuS-P(MEO2MA-co-OEGMA500)), 44 and in hybrid poly(Nisopropylacrylamide) (PNIPAm)-based nanogels decorated with plasmonic hollow gold nanoparticles (HGNPs-PNIPAm) loading bupivacaine. 45 At 0.1 mg/mL, these prior works showed cell viability percentages lower than those displayed in the present work, probably owing to the presence of free bupivacaine, HGNPs, or CuS nanoparticles, except for macrophages 37 and U251MG. 45 Cell membrane damage was assessed by means of cell apoptosis studies developed by flow cytometry (Figures S3 and S5). The results obtained did not reveal remarkable differences between control and treated samples. Only BNCs treatment in fibroblasts and macrophages involved a slight increase (≤10%) in total apoptosis rate (early apoptosis + late apoptosis) and necrosis percentage, respectively. Furthermore, U251MG recorded an increase in the total apoptosis rate of 12−16% in regard to the control sample when bupivacaine hydrochloride or BNCs were present in the samples. These results are in accordance with our previous results related to cell apoptosis after treatment for 24 h with PNIPAm-based nanogels loaded with bupivacaine, 45 where free bupivacaine ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17225 treatment also involved a change in apoptosis, necrosis, and viability percentages in U251MG cells. The distribution of cell cycle phases was evaluated by flow cytometry (Figures S4 and S5). Cell treatment with BNCnanogels, BNCs, empty drug-free nanogels, and bupivacaine hydrochloride solution for 24 h did not exert significant changes in cell cycle, displaying changes in percentages up to 10% which is in agreement with previously reported results for bupivacaine-loaded HGNPs-PNIPAm nanogels. 45 However, a superior effect was observed in macrophages whose S phase was increased (3−23%) with a countervailing reduction in the G1 and G2 phases when cells were treated with the materials assayed showing a higher effect when nanogels were present in the sample. Therefore, cell treatment with these materials was not detrimental for DNA and cell nuclei as cell cycle was not significantly affected. Finally, endotoxin levels in BNCs and BNC-nanogels were quantified by means of the Pierce Chromogenic Endotoxin Quant Kit as mentioned before. The endotoxin concentrations obtained from these samples were much lower than the approximate threshold pyrogen dose for humans (0.05 ng/mL = 0.5 endotoxin unit (EU)/mL) for a nonintrathecal administration, as indicated by the FDA. 46 In conclusion, the synthesized thermoresponsive nanogels loaded with BNCs did not significantly affect cell metabolism, membrane, and cycle on the cell types studied, as well as it did not involve potential Figure 4. Sciatic nerve blockade effect for rats injected with free bupivacaine hydrochloride, nanogels embedding BNCs and BNC formulations. (a) Sensory nerve blockade using the hot plate test. Behavioral reflexes: (b) paw withdrawal test scores and (c) adapted extensor postural thrust scores. (d) Gait ability test scores. (e) Flexion contracture test scores. (f) Motor nerve blockade using weight bearing test. (g) Scheme showing the in vivo mechanism proposed for the nerve blockade using BNC-nanogel formulations. Score 1 represented a normal reflex or response, while score 4 denoted the absence of reflex or response. The nonexistence of contracture was assessed as 0 and as 1 if the foot showed a flexion contracture. Data are presented as mean ±SD (N= 4 per group) (*p< 0.05, **p< 0.01, ***p< 0.001, and ****p< 0.0001). ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17226 detrimental endotoxin levels in a potential in vivo administration, pointing to their promising biomedical application in peripheral nerve pain relief. In Vivo Sciatic Nerve Blockade. Rats were injected close to the sciatic nerve root with the drug formulations containing all of the same bupivacaine equivalent dose of 2 mg. The injected dispersions were: 43 mg/mL BNCs (94% bupivacaine loading), 47 mg/mL BNC-nanogels (85% bupivacaine loading), and 40 mg/mL bupivacaine hydrochloride solution. The sensory nerve blockade was evaluated using the hot plate test. Results in Figure 4a showed that all of the bupivacainecontaining injected formulations, BNC-nanogels, BNCs, and free bupivacaine hydrochloride, achieved the maximum sensory nerve blockade (thermal latency higher than 8 s) by the time of the first measurement, that is, 30 min after injection. The nerve blockade remained 80% after 2 h for the free bupivacaine hydrochloride administration, and the normal sensory nerve behavior was recovered between 4 and 6 h after treatment. In the case of BNCs, the sensory nerve blockade gradually decreased in the first 6 h reaching at that time the 10% of the maximum effect. However, results in Figure 4a indicated that BNC-nanogels produced a longer duration of sensory nerve blockade in comparison to the effect of the nonencapsulated BNCs and the one of the free bupivacaine hydrochloride solution. The sensory nerve blockade for BNCnanogels was effective for at least 8 h after injection, remaining at this time the 76% of the maximum nerve block. This outcome suggested that the OEGMA-based nanogels were able to retain the bupivacaine in the proximity of the sciatic nerve prolonging the anesthetic effect more than 8 h with the same dose of drug (2 mg) as the ones contained in the other formulations. Probably, this effect is attributed to the thermoresponsive character of the polymer, which, when reaching physiological temperature, becomes hydrophobic, shrinks, and packs the nanocrystals encapsulated in its interior acting as a diffusion barrier for the drug remaining isolated from the medium due to its hydrophobic nature and producing a sustained release. Obviously, there is no correlation between the in vitro (Figure 2g) and in vivo effect observed; therefore, the interaction of the material with the tissues plays a very important role in the prolonged duration of action observed. The evaluation of the two behavioral reflexes or responses pointed out the same trend as the sensory nerve block test. For the free bupivacaine hydrochloride formulation, the paw withdrawal tests showed a score of 4 (Figure 4b), with total absence of mechanical sensitivity for 1 h after injection. Also, for this formulation, the adapted extensor postural thrust analysis showed no flexor function in the animals, the presence of a flexor contracture in the treated hind paw, along with a total lack of mobility in the limb during the first hour (Figure 4c−e). Two hours after the administration of the free bupivacaine hydrochloride, the animals had almost recovered the paw withdrawal reflex, the flexor contracture in the treated hind paw was reduced, showing a half-open paw and they had recovered partially the movement of the leg. The only parameter that remained affected up to 4 h after administration was the flexor function that was totally recovered after 6 h of study. The BNCs showed anesthetic efficiency that lasted until 4 h of study (Figure 4a); however, differences can be observed in the behavioral responses and in the scores analyzed compared to the effect of the free bupivacaine hydrochloride solution. Animals treated with the BNCs showed a delayed mechanical sensitivity reflex and a weak grip (scores between 2 and 3 in Figure 4b,c), but the reflexes were not totally impeded. The animals showed a slight hindered movement of the limb (scores between 1.5 and 2.3 in Figure 4d) and a slight flexor contracture in the treated hind paw (Figure 4e). Therefore, the functionality of the limb was not totally lost as in the case of bupivacaine hydrochloride solution. Finally, the effect of administrating BNC-nanogels was different; they exhibited a more prolonged anesthetic effect with an extended duration of action persisting for more than 8 h (Figure 4a). Again, the motor function of the leg was not completely hindered, as the behavioral responses showed a slight response to paw withdrawal and the adapted extensor postural thrust analyses (Figure 4b,c). Also, the movement was slightly hindered (scores between 2 and 3 in Figure 4d) and a flexor contracture in the treated foot was somehow detected during 8 h(Figure 4e). These findings confirmed the prolonged effectiveness produced by the encapsulation of BNCs into nanogels. Saline controls were also performed, and no anesthetic effect was found (results not shown). Motor nerve blockade was analyzed for the different formulations using the weight bearing test. Results in Figure 4f showed a slight influence on motor block for BNCs and BNC-nanogel formulations that is compatible with the preservation of some reflexes in the limb, with exception of bupivacaine hydrochloride solution during the first hour of administration, where there was a superior motor nerve blockade. Previous investigations using bupivacaine-loaded PLGA microparticles to produce sciatic nerve blockade 47 did not achieve the maximal nerve block by the first 30 min although they administrated 38.5 mg of bupivacaine hydrochloride. This initial onset might be attributed to the slow degradation of PLGA by hydrolysis of the ester bond present in the backbone, facilitating a controlled release of encapsulated cargoes. These PLGA microparticles achieved a duration of sensory block of more than 11 h, providing a prolonged duration of the local anesthesia. Other authors 2 reported a nerve block duration of 6 h using PLGA microparticles loaded with a total dose of 50 mg of bupivacaine hydrochloride. Other formulations reported in the literature include bupivacaine-loaded liposomes, 48 which reached a duration of sciatic nerve block of 7.3 h, upon administration of liposomes containing 6 mg of bupivacaine. Our formulation achieved a continuous extended nerve blockade, for more than 8 h, with a total dose of only 2 mg of nanocrystallized bupivacaine. The hydrophobicity of bupivacaine free base probably facilitates its pass through cell membranes and also binds strongly to the hydrophobic lipid bilayer, thereby protecting molecules from diffusion to extracellular media and to the bloodstream, reducing their clearance in comparison to the ionized form (hydrochloride salt). 49,50 Probably bupivacaine being a weak base diffused in its unionized form from the BNC-nanogel formulations to the interior of the cell, which ionized due to the slight acidic interior environment blocking the inner surface of the voltagegated sodium channels avoiding action potential to propagate. Moreover, this extended duration of blockage is probably also explained by the immobilization of BNC-nanogels in the injection site around the sciatic nerve due to their hydrophobic character, preventing particle diffusion and fast drug clearance. At 37 °C, the nanogels are partially collapsed to half of their volume (Figure 1g), giving a transition to a more hydrophobic structure reducing their elimination from the body, interacting efficiently with the tissues and with the afferent nerve fibers. The hydrophobic character of the partially collapsed nanogel ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17227 could retain the formulation in the injection site maintaining drug levels in the therapeutic window, which explains the different effect between nonencapsulated and encapsulated BNCs. A schematic illustration of the mechanism proposed for the regional anesthesia effect obtained for BNC-nanogels is depicted in Figure 4g. Other authors reported the immobilization of PLGA microparticles (MPs) embedded in thermosensitive macrogels based on PLGA−PEG−PLGA to obtain high local drug concentration for long-acting analgesia. 51 Those PLGA MPs were prepared using oil-in-water (o/w) emulsification method for the encapsulation of bupivacaine free base. PLGA MPs containing 40 mg of bupivacaine were administrated embedded in a PLGA−PEG−PLGA solution having a sol−gel transition when exposed at body temperature (37 °C). They achieved a 22 h block of sciatic nerve, which is more than twice the duration accomplish by our BNCnanogels, but using 20 times more amount of drug. In our formulation, we used nanocrystallized bupivacaine, which produced sustained release and enhanced the long-term nerve block. In general, for several applications, it is desirable to obtain a longer duration of sensory block than motor block, for instance, in obstetric anesthesia where the mother should maintain the motor functionality while still obtaining pain relief. In the case of peripheral nerve pain treatment, something similar occurs; it is desirable for sensory block to be of longer duration than motor block to relieve the pain but not resulting in a paralyzed limb. Using BNC-nanogels, we have obtained still mobility in the limb causing sensory nerve block enough to relieve the pain. The amount of myelin around the nerve axons is different in afferent (i.e., nociceptive sensory fibers innervated in the direction toward the bone marrow) than that in efferent (i.e., motor neurons that exit the bone marrow innervating the skeletal muscles) nerve fibers; therefore, a specific anesthetic dose is able to block the nerve impulse transmission in one direction (sensory block) while allowing the motor neurons to carry efferent impulses to the effector in the other direction, which results in movement. Another important aspect to take into account is the lack of observed in vivo toxicity exhibited after the treatment with BNC-nanogels. Other polymeric bupivacaine-loaded delivery systems have reported local toxicity, myotoxicity, and inflammation probably attributed to a fast release kinetics of bupivacaine (burst release), and especially to the accumulation of polymeric residues that remain attached to the tissues. 52−54 The in vivo sustained-release kinetics of the BNC-nanogels among with the excellent biocompatibility of PEG-based nanogels are the reasons for the low toxicity exhibited by these nanomaterials. Therefore, BNC-nanogels can be potentially used in postoperative pain control by local infiltration, for instance, in mammoplasty, total knee arthroplasty, hemorrhoidectomy, and inguinal hernia repair. BNC-nanogels are excellent candidates for epidural uses as they allow motor functionality while obtaining pain relief. In this sense, they Figure 5. Tissue sections of the sciatic nerve root area collected 4 days post-inoculation (dpi): (a−c) Rat injected with bupivacaine nanocrystals (BNCs) nanogels. No significant changes in nerve, muscular, and adipose tissues are observed. (a) Hematoxylin−eosin (HE) 4×, (b) Masson′s trichrome 10×, and (c) Luxol fast blue 10×;(d−f) rat injected with BNCs. Mild to moderate lesions around the injection site including fibroplasia and muscular fiber regeneration. (d) HE 4×, (e) Masson’s trichrome 4×, and (f) Luxol fast blue 4×;(g−i) rat injected with free bupivacaine hydrochloride. No significant changes are observed. There is a mild fibrous reaction along the injection site (arrow). (g) HE 4×, (h) Masson’s trichrome 10×, and (i) Luxol fast blue 10×. ACS Applied Materials & Interfaces www.acsami.org Research Article https://doi.org/10.1021/acsami.1c00894 ACS Appl. Mater. Interfaces 2021, 13, 17220−17235 17228 (53) Kohane, D. S.; Langer, R. Biocompatibility and Drug Delivery Systems. Chem. Sci. 2010,1, 441−446. (54) Padera, R.; Bellas, E.; Tse, J. Y.; Hao, D.; Kohane, D. S. Local Myotoxicity from Sustained Release of Bupivacaine from Microparticles. 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