Nanoengineering palladium plasmonic Nanosheets inside polymer nanospheres for photothermal therapy and targeted drug delivery

www.afm-journal.de 2106932 (1 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH ReseaRch aRticle Nanoengineering Palladium Plasmonic Nanosheets Inside Polymer Nanospheres for Photothermal Therapy and Targeted Drug Delivery Laura Uson, Cristina Yus, Gracia Mendoza, Eric Leroy, Silvia Irusta, Teresa Alejo, David García-Domingo, Ane Larrea, Manuel Arruebo,* Raul Arenal, and Victor Sebastian* The incorporation of plasmonic nanoconstructs in biodegradable polymeric nanoparticles (NPs), together with therapeutic drugs in a controlled procedure is of interest for different applications in Nanomedicine. Advanced hybrid nanomaterials can be engineered by combining the in situ formation of plasmonic palladium nanosheets (NSs) and the proper ionic nature of the encapsulated drug. This study presents a new procedure to synthesize hybrid nanostructures by a Pickering double emulsion. Anisotropic palladium (Pd) NSs with unique near-infrared (NIR)-optical properties can be assembled within a poly lactic-co-glycolic acid matrix of <200nm NPs, when Pd precursors are in situ reduced via a gas-phase procedure. The hybrid nanomaterials respond to external NIR light stimulus. The unprecedented precision to assemble, in a single stage, plasmonic nanoconstructs having a total loading selectivity, when encapsulated in combination with hydrophobic drugs, offers new opportunities in the new class of theragnostics, in particular when triggered drug delivery and photothermal therapies are required. DOI: 10.1002/adfm.202106932 L. Uson, C. Yus, S. Irusta, T. Alejo, M. Arruebo, R. Arenal, V. Sebastian Instituto de Nanociencia y Materiales de Aragón (INMA) CSIC-Universidad de Zaragoza Zaragoza 50009, Spain E-mail: [email protected]; [email protected] L. Uson, C. Yus, S. Irusta, T. Alejo, A. Larrea, M. Arruebo, V. Sebastian Department of Chemical Engineering University of Zaragoza Campus Río Ebro-Edificio I+D C/ Poeta Mariano Esquillor S/N Zaragoza 50018, Spain and the toxicity and resistance associated to the drugs used, such as chemotherapy medicines, demand the design of more effective, safe, and reliable drug delivery vectors.[1] Polymers are of interest in therapeutic applications because exhibit a great synthetic versatility to customize the targeted application and satisfy the stringent requirements of the regulatory authorities.[2] Polymers are engineered into the nanoscale[3] to circumvent some of the risks and disadvantages associated with traditional drug administration routes by controlling:[4] 1) the drug payload, 2) pharmacokinetics and release in the site of action, and 3) the internalization through biological membranes. Then, polymer nanoparticles (NPs) enable maintaining the therapeutic efficacy and the active targeting to the specific site needed of therapeutic action. Polymer NPs can be designed to improve drug bioavailability (i.e., the portion of the bioactive compound that reaches systemic circulation and performs the therapeutic functions), by: 1) increasing drug absorption through enhanced solubility, this is especially important for hydrophobic drugs[5] or The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.202106932. 1. Introduction Nanomedicine and the wide variety of nanomaterials designed up to date have the potential to enable novel modalities for diagnosis and therapy. The complex treatment of several diseases © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. L. Uson, G. Mendoza, S. Irusta, T. Alejo, M. Arruebo, V. Sebastian Networking Research Center on Bioengineering Biomaterials and Nanomedicine CIBER-BBN Madrid 28029, Spain G. Mendoza, D. García-Domingo Aragon Health Research Institute (IIS Aragón) Zaragoza 50009, Spain E. Leroy Université Paris-Est Institut de Chimie et des Matériaux Paris-Est UMR 7182 CNRS – UPEC 2 Rue H. Dunant, Thiais 94320, France R. Arenal, V. Sebastian Laboratorio de Microscopías Avanzadas Universidad de Zaragoza Zaragoza 50018, Spain R. Arenal ARAID Foundation Zaragoza 50018, Spain Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (2 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH 2) by facilitating the diffusion through biological membranes.[6] Drug release must be controlled and maintained at therapeutic levels, by adjusting the composition of the polymer matrix used as drug carrier and the drug loading. For this purpose, it is necessary to control several parameters in the polymeric NPs used as drug carriers: their chemical composition, size, polydispersity, architecture, surface chemistry, including hydrophilicity, hydrophobicity, and charge density.[2] Compared to other drug vectors, such as liposomes, polymeric NPs allow increasing the stability of carried drugs and control their release due to the possibility of adapting their size, structure, and chemical composition.[7] Poly lactic-co-glycolic acid (PLGA) is a widely used polymer in drug delivery due to its biodegradability and demonstrated biocompatibility.[8] PLGA is the most common synthetic biopolymer approved by the Food and Drug Administration (FDA) and the European Medicine Agency in drug delivery systems and devices because it allows a sustained drug delivery thanks to its controlled hydrolytic degradation and the encapsulation of hydrophilic and hydrophobic therapeutic molecules.[9] Furthermore, PLGA commercially exists in different compositions to modulate its degradation kinetics and its properties are easily modifiable using site-specific chemistry in its structure.[10] PLGA can be self-assembled into micro or nanospheres by using different synthetic procedures including emulsionsolvent evaporation, nanoprecipitation, spray drying, solvent displacement, salting out, etc. The combination of biocompatible polymers and metal NPs as hybrid nanostructures can perform several functions other than drug delivery, emerging as promising combination vectors in the new class of theragnostics used in highly sensitive biodetection,[11] medical imaging,[12] and triggered drug delivery.[13] Metal NPs can be either incorporated into the inner NP space or attached on the surface of the nanoentity.[11a] This type of hybrid NPs are usually produced by complex multistage procedures based on the covalent and noncovalent grafting to polymers of preformed metal NPs.[14] Although some of the reported procedures based on those previous strategies are easy to implement,[15] the loading control of metal NPs is poor and its reproducibility limited.[14b] It should be highlighted that the majority of nanomaterials targeted to medical applications cannot progress to clinical stage or to the market because large-scale manufacturing is in many cases not feasible due to complex synthesis procedures and the lack of reproducibility.[2] Consequently, our group developed in 2016 a novel procedure to circumvent the weaknesses of some of the previous production techniques.[14b] This procedure was based on the in situ reduction of metal ions loaded in the internal aqueous phase of a water-oil-water (w/o/w) polymeric double-emulsion of PLGA. After the emulsification process, the ions were reduced on demand by the citrate ions present in the emulsion using a redox process activated by temperature at soft conditions (40°C)topreserve the polymer properties. Following this procedure, a tunable payload of spherical gold (Au) NPs (size ≈ 10nm) could be encapsulated in each PLGA NP (size ≈ 175nm).[14b] A further development was carried out by developing a continuous, robust, and scalable process to prepare these hybrid metalpolymeric NPs by a microchannel emulsification process.[16] In this case, a three-stage process in continuous flow was designed; the first two stages were aimed at the production of the w/o/w PLGA double-emulsion and the loading of the reducing agent, as well as the Au precursor. Finally, the third stage was devoted to supply for 10 min the thermal energy required to activate the redox process. This procedure was able to supply a continuous production of Au-loaded PLGA hybrid NPs of 2.8 mg s−1.[16] Although the payload of Au NPs by the reported procedure was totally selective to the encapsulating PLGA NPs (100% loading efficiency), the shape of the inner Au NPs was not modulated. This issue hinders the applicability of the resulting hybrid NPs produced by the in situ procedure to further uses in nanomedicine where metal NPs of specific anisotropic shapes not only can be used as contrast agents[17] but also as therapeutic agents by transducing optical energy into thermal energy.[17a] This optical properties are attributed to a collective oscillation of electrons in the conduction band of metal NPs, this oscillation is named surface plasmon resonance (SPR),[18] and depends on NPs chemical composition, size, interparticle distance, shape, and aspect ratio.[19] Plasmonic NPs can emit light (i.e., electromagnetic radiation used in imaging applications) or absorb light and dissipate it in form of heat (applied in hyperthermia treatments).[20] Water molecules and tissues have reduced light absorption at near-infrared (NIR) wavelengths. In this case, coupling the NIR plasmon resonance absorption of some metal NPs into thermal energy has been exploited to photothermally destroy malignant cancer cells.[21] To date, only NIR sensitive metal NPs were loaded into large biodegradable polymeric microstructures,[11b] and thermoresponsive polymeric nanostructures,[13,22] but not in biodegradable nanostructures (having faster degradation kinetics, and reduced immunogenic reaction than the reported ones). These facts deemed important because size, shape, composition, and surface chemistry of nanovectors influence their behavior in biological contexts. For instance, the use of thermoresponsive polymers such as Poly(Nisopropylacrylamide)-PNIPAM could be a concern in terms of bioaccumulation and toxicity.[23] Vectoring in the microscale or nanoscale is also crucial, since nanomedicine applications generally demand nanovectors with reduced bioaccumulation and no immunogenicity. For instance, it has been shown that nanovectors smaller than 30nm can diffuse into systemic circulation following administration into the lungs.[24] Then, the design of a simple procedure able to selectively load NIR sensitive NPs into a biopolymeric matrix, such as PLGA, at the nanoscale should be deemed important as a theragnostic nanotool. We present here a novel and simple method to embed NIR sensitive palladium (Pd) metal nanosheets (NSs) of 1.5 nm thickness in PLGA NPs by an in situ production process. This novel approach is based on the gas-phase reduction of the metal ions loaded in the polymeric NPs by a reactive gas atmosphere with a dual role: 1) reduction agent and 2) capping agent to modulate the shape of metal nanostructures. The solubility of gases is generally limited in liquid media, having the benefit of leaving the reaction media at atmospheric conditions,[25] without leaving any cross-contamination that requires an extra purification process. On the other hand, the use of reducing agents added either as a payload in the emulsification media[14b] or in a post-treatment process[16] has evidenced some difficulties related to the emulsion formation and the nucleation/growth events of metal NPs; and even could irreversibly react with the bioactive encapsulated compounds. Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (3 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH Pd nanostructures have emerged as important materials in the field of nanomedicine due to their remarkable chemical[26] and optical properties.[27] Pd NSs have strong optical absorption in the NIR region and high photothermal conversion efficiency (52%) at 808 nm,[28] so they are excellent candidates as drug-release activation agents and to facilitate the induction of local hyperthermia.[27] On the other hand, many anisotropic Au nanostructures exhibiting NIR properties can be faded upon irradiation with high-power NIR lasers. This phenomenon occurs because the heat generated after NIR irradiation induces reshaping in the anisotropic structure, leading to the loss of the phothothermal properties of the NPs and thereby inevitably imposing limitations in their practical therapeutic applications.[17b] Pd nanostructures, with a bulk melting point higher than that of Au (MPPd = 1,828 K vs MPAu = 1,337 K) have been used to address this limitation.[17c] Pd nanosheets with a thickness of 1.5 nm were assembled in this work inside of PLGA NPs with unprecedented precision and with a 100% loading efficiency. The incorporation of metallic NPs that respond to external stimuli such as NIR light, allows the creation of triggered drug delivery systems that upon activation can provide precise control over time, dose, and location of drug administration. In addition, in this work the photothermal effect induced by NIR SPR absorption was investigated as a therapeutic nanotool. Integrating Pd NSs, as plasmonic nanomaterials, with polymers can improve the biocompatibility, biosafety, and blood circulation half-life of these nanostructures.[29] Pd NSs heating can cause deformations in the PLGA matrix, which vitreous transition temperature range between 45 and 50°C,[30] and could induce the release of the encapsulated drug on demand.[31] Currently, the most common activation systems are those that use UV–VIS light, which require a large amount of applied energy, which allows activating the administration systems by modifying their chemical structures. However, this light is phototoxic (UVB, UVC) and has low penetration into the tissues, which only allows its use near the surface.[32] As an alternative, the emergent option is the radiation in the NIR (700–1000 nm), range in which the majority of the absorbers in living systems show minimal absorption.[33] In addition, it has less delivered energy, so it is less harmful to cells. Therefore, the hybrid NPs produced in this work were endowed with a triple functionality to: i) Improve drug solubility and bioavailability, ii) modify the drug release on demand at the point of action, and iii) achieve local hyperthermia, due to NIR light absorption, that produces localized cytotoxic heat able to ablate cells. In this work, the drug encapsulation study was carried out using bupivacaine, a known anesthetic molecule that at low pH is protonated, and therefore has a hydrophilic character (pKa 8.4), whereas if the pH is increased, it becomes more hydrophobic.[34] The release mechanism of PLGA/Bupivacaine systems was previously studied,[35] although the proposed hybrid nanosystem allows in addition a sustained release provided by the biodegradation of PLGA as well as the fast supply of drug on demand though a NIR laser activation and local hyperthermia. Bupivacaine was selected as a model drug being one of the most common local anesthetics used in peripheral nerve block. Most of the prolonged duration nanoparticulated carriers containing local anesthetics (such as bupivacaine) produce a constant release profile providing a continuous extended nerve blockade without allowing for changes in the patient’s daily physical activity or level of pain relief. In most of those systems the anesthetic release does not cease until all the payload is depleted. To overcome such limitation, triggerable drug delivery systems, as the one here reported, have been developed. 2. Results and Discussions 2.1. Production of Hybrid Pd-PLGA NPs Pd NSs have been prepared in previous studies[36] using organic capping agents (i.e., cetyltrimethylammonium bromide-CTAB) to promote the anisotropic growth of Pd nanocrystals achieving the NIR absorption properties required. However, the presence of CTAB during the emulsification process, as a quaternary ammonium surfactant, can alter the w/o/w emulsion formation, besides CTAB is quite toxic to cells at sub-micromolar doses.[37] Thus, considering some of our previous results of Pd NSs growth without CTAB,[38] the synthesis of Pd NSs was adapted to a low temperature process to avoid PLGA emulsion destabilization. Pd NSs were first produced at 30°Cassisted by the capping effect of Br− ions to the (100) planes and under a pressurized atmosphere of CO (see experimental section) to increase the CO solubility in the media. The presence of CO is critical because it can supply electrons to reduce Pd2+ ions to Pd0 atoms and prevents the growth of Pd crystals along (111) because CO strongly adsorbs on basal (111) planes of Pd nanosheets.[36a] Figure1a,b corresponds to representative transmission electron microscope (TEM) images of Pd NSs produced under these synthesis conditions where nonregular hexagonal shape nanocrystals of around 42± 5nm were observed. Lattice fringes with an interplanar distance of 0.23nm, corresponding to which can be ascribed to metallic Pd (111) surfaces,[39] can be observed in the high-resolution transmission electron microscopy (HRTEM) image displayed in Figure1c. This is in agreement with previous TEM studies of Pd NSs and suggests that the nanocrystals are bound by two (111) basal planes.[36] The presence of stacked nanosheets (see Figure 1d) enables thickness measurement of the ultrathin structure, resulting in a mean thickness of 1.5 nm, which corresponds to less than 9 atomic layers. The ultrathin morphology of Pd NSs is crucial for their specific optical properties in the NIR region[36a] (Figure1j). As a challenge, the growth of Pd NSs was translated to the interior of PLGA NPs. The most widely used method for the synthesis of PLGA NPs is based on an emulsification process, to form the micelles of PLGA NPs, following by the evaporation of the organic solvent at room temperature to precipitate PLGA into a NP since PLGA is not soluble in water.[40] Depending on the nature of the drug to be encapsulated, simple emulsion (oil-water, o/w) for hydrophobic molecules,[41] or double emulsion (w/o/w)[42] for hydrophilic ones are commonly used. Herein, the Pd NSs reagents (hydrophilic) were loaded in the internal water compartment of the w/o/w micelles, being separated from the continuous aqueous phase by the organic layer of acetyl acetate, where PLGA is dissolved (Figure2a). Sodium cholate hydrate was selected as surfactant in the w/o/w interface. Then, the Pd NSs reagents were confined in Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (4 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH micelle like nanoreactors where the nucleation and growth events would proceed as soon as an electron donor was introduced to transform high soluble Pd ions into nonsoluble Pd atoms. The emulsion was introduced immediately after its formation in a CO-pressurized autoclave and was kept at 30°Cfor 40min (see the Experimental Section). The ethyl acetate layer in the micelles should not be evaporated before CO treatment because its evaporation would promote the release of Pd2+/ Br− ions to the continuous aqueous phase, precluding the proper assembly of Pd NSs inside PLGA NPs (Figure2a). Figure1e depicts a representative scanning electron microscope (SEM) image of PLGA NPs where Pd NSs where assembled by this novel and simple procedure. Particle size histograms determined by dynamic light scattering show that the size of PLGA NPs slightly increased when Pd NPs were embedded in their interior, (182± 62nm without Pd and 197 ± 43nm with Pd loaded NSs, respectively) (Figure S1a,b, Supporting Information). The inset in Figure1e also shows the hue of the emulsion before and after CO treatment. The Pd-Br complex shows the typical orange color, whereas the presence of Pd NSs turns the emulsion into a dark bluish hue. It should be highlighted that Pd NSs can be formed in only 150 s when the Pd2+ reduction is performed in a microfluidic system at high temperature (150°C),[36b] butin this case a large reduction time was required (40min) because the process occurs at low temperature (30°C)andCO should diffuse through several water/organic layers that conform the w/o/w PLGA micelle (Figure 2). Figure 1f shows a representative high-angle annular dark-field scanning TEM (high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM)) image of PdPLGA NPs, where it is depicted that Pd NSs grow selectively only in the interior of the PLGA NPs. Due to the z-contrast information provided in HAADF images,[43] Pd NSs were observed with a Figure 1. a) TEM image of Pd NSs produced with CO pressure of 6bar after 40min at 30°C. b) High magnification image of a Pd NS to show its hexagonal structure. c) HRTEM image of a Pd NS showing lattice fringes, the d-spacing corresponds to Pd (111). d) Stack of Pd NSs located perpendicular to the TEM grid. e) SEM image of PLGA NPs loaded with Pd NSs. Inset, optical images of the Pd-PLGA emulsion before and after the gas-phase CO treatment. f) Representative HAADF-STEM image of a PLGA NP loaded with Pd NSs to show the selective growth of Pd inside PLGA NPs and no in the interparticle space. g,h) High magnification HAADF-STEM images of Pd-PLGA NPs to observe the spherical assembly of Pd NSs. I) Energy-dispersive X-ray spectrum of a Pd-PLGA NPs to determine the presence of Pd inside PLGA NPs. j) UV–vis NIR Spectrum of Pd NSs dispersed in water. Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (5 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH brighter contrast than carbon from PLGA. Figure1g,h shows high magnification HAADF-STEM images of some representative Pd-PLGA NPs, where it is clearly observed that Pd hexagonal and triangular NSs are assembled in the interior of PLGA NPs having an unprecedented spherical pattern of assembly. Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of Pd atoms (Figure 1i). Besides the need of using CO to control the reduction of Pd2+ and the anisotropic growth of Pd NSs, the presence of Br− ions is critical to produce a homogenous nanosheet morphology. Indeed, the absence of Br− ions in the micelle during the emulsification process produces a combination of nanowires and heterogenous NSs (Figure S2, Supporting Information). This result is in agreement with previous works,[36a] where the presence of Br− ions was justified because they regulate the lateral growth rate of Pd NSs thanks to their selective binding to the (100) planes. 2.2. Production of Hybrid Bupivacaine-Pd-PLGA NPs Considering that Pd NSs were successfully embedded in the interior of PLGA, the following step was to encapsulate a therapeutic drug without modifying the growth of Pd NSs and keeping the NIR sensitive functionality (Figure 2b). Ionized and unionized bupivacaine forms were loaded during the emulsification process (see the Experimental Section) following some of our previous protocols.[44] Unionized bupivacaine-uiBup (bupivacaine free base), due to its hydrophobic nature, was loaded in the organic phase of the w/o/w emulsion together with the dissolved PLGA polymer (uiBup-Pd-PLGA). Contrarily, ionized bupivacaine-iBup (bupivacaine hydrochloride), with a more hydrophilic nature than the bupivacaine free base, was loaded in the internal aqueous compartment of the w/o/w micelle, together with the Pd reagents (iBup-Pd PLGA). This step is also critical because the drug could alter the emulsion stability or even block the growth of metal NPs because it could be strongly adsorbed on Pd nanocrystal facets hindering the specific CO/Br− adsorption or the growth. Pd-PLGA NPs without drug showed a negative zeta potential value (−30.28± 3.2mV) at neutral pH, whereas uiBup-Pd-PLGA NPs loaded with unionized bupivacaine showed positive values (21.43 ± 2.9 mV). The negative charge of pure PLGA NPs could be attributed to the presence of ionized carboxyl groups of sodium cholate surfactant on the PLGA NP surface.[45] However, the positive charge of uiBup-Pd-PLGA NPs might be Figure 2. a) Scheme of the synthesis process to assemble selectively Pd NSs in PLGA NPs by a w/o/w emulsification process, assisted by a CO treatment and solvent evaporation. b) In situ encapsulation of unionized bupivacaine and Pd NSs to yield NIR triggered drug delivery systems. Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (6 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH due to the neutralization of the polymer charge by electrostatic interaction with the drug, and residual adsorbed bupivacaine that covers the PLGA NPs surface.[13] Particle size histograms determined by dynamic light scattering (DLS) show that the size of Pd-PLGA NPs does not increase when unionized bupivacaine was encapsulated (185± 31nm) (Figure S1c, Supporting Information). The analysis of SEM images of uiBup-Pd-PLGA NPs determined a mean particle size of 146 ± 56 nm, which is slightly smaller than the one obtained by DLS (Figure S3a, Supporting Information). The particle size difference observed between SEM and DLS techniques was expected and it is in agreement with the literature,[46] since PLGA-Pd NPs in DLS measurements are solvated while during SEM they are dried. Fourier transform infrared spectroscopy (FTIR) studies (Figure S4, Supporting Information) were performed in order to confirm the presence of bupivacaine into PLGA NPs and the existence of possible interactions with the polymer. The FTIR spectrum of unionized bupivacaine (Figure S4a, Supporting Information) shows the absence of OH stretching band at 3508 cm−1, characteristic of the monohydrate drug form and the shift of the absorption band at 3243 cm−1, assigned to NH stretching vibration to lower wavenumbers confirming the unionized state of the drug.[47] FTIR spectrum of bupivacaine loaded particles shows the PLGA characteristic absorption bands and also the most intense peaks of the drug (Figure S4b, Supporting Information). The small signal at 3508 cm−1 could indicate some protonation degree of the bupivacaine after the incorporation into the particles. Comparing loaded and unloaded particles spectra, a shift for the characteristic CO stretch (1756 cm−1) of the sodium cholate to lower wavenumbers can be observed in the uiBup-Pd PLGA spectrum.[48] On the other hand, the band associated to the presence of free carbonyl groups in bupivacaine (1647 cm−1) shifts to higher wavenumbers when loaded in PLGA particles. These facts suggest some interaction between these groups of bupivacaine with PLGA. Notice that in loaded and unloaded NPs, amide II bonds remain in the same position in the bupivacaine spectrum. Microwave plasma-atomic emission spectrometer (MP-AES) measurements determined that palladium loading in uiBupPd PLGA NPs corresponded to 17± 1.9wt%. That is, the concentration of Pd in the sample was around 6 × 10−4mg Pdper mg NPs. On the other hand, the determination of bupivacaine encapsulated in the uiBup-Pd PLGA NPs by gas chromatography-mass spectrometry (GC-MS) resulted in an efficiency of encapsulation of 19.1± 3.5wt% and the drug loading was 7.6± 1.8 wt%. This value is in the same order that previous publications where bupivacaine was loaded in NPs,[44,49] but in this case the additional assembly Pd nanosheets in their interior endow the PLGA NPs with an additional theragnostic nanotool. Regarding the iBup-Pd PLGA NPs where ionized bupivacaine was loaded in the aqueous compartment (Figure 2b), the NPs size was not substantially modified in comparison with that of PLGA NPs with Pd NPs and unionized bupivacaine, obtaining by DLS a mean size of 175± 42nm (Figure S1, Supporting Information). On the other hand, the surface charge (10.30± 0.8mV) is slightly lower than the one obtained for the NPs loaded with unionized bupivacaine (21.43 ± 2.9mV).This fact should be related to a limited bupivacaine loading achieved in PLGA NPs when the ionized form was used. In agreement with this insight, the ionized bupivacaine quantification by CG-MS was not as high as that with the unionized type, resulting an encapsulation efficiency of 5.52 ± 1.2 wt% and drug loading as low as 0.8± 0.2wt%. The different bupivacaine payload could be closely related with the high water-solubility of bupivacaine hydrochloride (ionized form) in comparison with the bupivacaine free base (unionized form). This is a key issue to consider in the solvent evaporation stage of PLGA NPs production, where the organic solvent release enables the polymer precipitation but also drug diffusion, reducing the encapsulated amount. In addition, the low affinity of small molecular weight hydrophilic/ionized drugs with the polymer can result in a low entrapment efficiency and a limited drug loading.[50] The simultaneous loading of iBup and uiBup was also attempted, but the loading results did not overpass the one produced with uiBup and it was not further considered. Figure3 depicts representative electron microscopy images of Pd-PLGA NPs encapsulating either unionized (Figure 3a,b) or ionized bupivacaine (Figure3c,d). It can be observed that the assembly of Pd NSs in presence of unionized bupivacaine is similar to the case where no bupivacaine was used (Figure1). However, the geometrical shape of Pd NSs was more heterogeneous than that without bupivacaine. It could be rationalized by the low solubility of unionized bupivacaine in water (40mgL−1),[51] favoring the mere interaction with Pd NSs facets during the growth process. Regarding the assembly of Pd NSs when ionized bupivacaine was encapsulated, Figure3c,d shows that both the dimension and shape, as well as the distribution of the NSs, were substantially modified regarding previous commented scenarios. This result is a clear evidence that ionized bupivacaine is interacting during the NSs growth, evidencing the challenge of growing nanocrystals under the presence of some drugs and the importance of selecting the proper loading scenario in the production of hybrid nanomaterials. In this case, loading the drug in a different compartment from the Pd precursor seems to avoid conflicts during the nucleation/growth events of Pd NSs. Then, Pd-PLGA loaded with unionized bupivacaine (uiBup-Pd PLGA) was selected for further analysis and for its application in triggered drug release and optical hyperthermia. 2.3. 3D Structure and Assembly of Hybrid Bupivacaine-Pd-PLGA NPs To shed light on the 3D distribution of Pd NSs in the hybrid uiBup-Pd PLGA NPs, we have performed electron tomography under cryogenic conditions. Indeed, these are the most appropriated conditions to work on polymeric nanosystems as these ones, which are very sensitive to the electron beam and the damage would avoid extracting such information.[52] Thus, cryo-electron tomography at −170°Chas been recorded on this system. The images from the tilt series (Figure3h–j; and Movie S1, Supporting Information) show the spherical morphology of the PLGA-bupivacaine polymer and the 3D distribution of the Pd NSs on this polymer. They confirm that the Pd NSs are located at the shell of the PLGA NP. This finding is not reported yet in the in situ production of hybrid nanomaterials at the nanoscale level and is very interesting since the Pd precursors were loaded at the core of the NP using a double emulsion approach (Figure2). Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (7 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH Pd nanosheets produced at the core of the w/o/w double emulsion could segregate at the interface of immiscible ethyl acetate-water interphase. This segregation would be governed by a decrease in interfacial energy as driving force. The Pd nanosheets get adsorbed onto the organic-aqueous interface and hence, the residual interfacial energy of the interfacial polymeric molecules would be released.[53] Due to the anisotropic structure of Pd NSs and the physisorption nature of this process, formation of Pd NSs monolayer at the interface is favored by the reduction in surface energy.[53] This mechanism has been well-documented in emulsion-related literature as Pickering emulsions.[53] However, this is the first time that a Pickering double emulsion is prepared at the nanometer scale without the need of first engineer the inorganic nanoconstructs before their loading in the emulsion. The self-assembly of nanoparticles in 3D has been only developed in colloidal microcapsules because at the submicrometer scale, the nanoscale particles are characteristically unstable at the interface due to thermal disorder.[54] Consequently, the achieved results are remarkable and the proposed new procedure for hybrid nanomaterials production is a seminal route to nanoscale assemblies. 2.4. Photothermal Properties: Light Triggered Drug Delivery and Thermal Ablation The photothermal properties of uiBup-Pd PLGA NPs induced by the unique NIR SPR absorption of Pd NSs, considering that Figure 3. Electron microscopy images of Pd-PLGA NPs loaded with: a,b) unionized bupivacaine and c,d) ionized bupivacaine. Insets, optical images of the Pd-PLGA-Bupivacaine emulsions before and after the gas-phase CO treatment. e) Photothermal characterization. Temperature variation under 808nm laser irradiation (1.8 Wcm−2, 2.4mgmL−1 of NPs) for 5min. Heating rate of Pd-PLGA NPs loaded with unionized bupivacaine, PLGA NPs without Pd (control 1) and water (control 2). f) Photothermal stability of Pd-PLGA bupivacaine NPs during 10 successive irradiation cycles with heating from 37to 45°C. g)SEMimage of Pd-PLGA bupivacaine NPs after 10 successive irradiation cycles. h–j) TEM images of a uiBup-Pd PLGA-bupivacaine at −67°,0°,and +67°relative to the electron beam. Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (8 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH Pd only when having sheet-like morphology is able to display its characteristic SPR absorption in the NIR region of the electromagnetic spectrum, was studied by monitoring the temperature of an aqueous solution (1 mL, 2.4 mg mL−1 of NPs) irradiated by a NIR laser (808 nm, 1.8 Wcm−2). As it can be observed in Figure 3e,f, a dispersion of uiBup-Pd PLGA NPs with a content in Pd of 1.45µgmL−1 increases the temperature from room temperature conditions (25°C)to45°Cin 5 min after irradiation. The resulting heating properties are attributed to the SPR of Pd nanosheets absorbing the NIR electromagnetic radiation in an efficient manner. In comparison, the temperature of the dispersion in absence of NPs or Pd NSs increased only 5°C(Figure3e,f) due to the particle scattering and the reduced energy absorption of water and PLGA in the NIR region.[27] The efficient transduction of light into heat was therefore preserved even after encapsulating the light-absorbing Pd NSs within the polymeric matrix. The stability of the NPs under laser irradiation was confirmed by performing ten successive irradiation cycles (Figure3e,f). The suspension cooled down to body temperature in only 3min after turning the light off, which makes these NPs potentially interesting for pulsatile photothermosensitive applications as triggered drug delivery vectors. SEM images were analyzed to evaluate if NPs morphology had changed. Figure 3g; and Figure S3 (Supporting Information) shows that the uiBup-Pd-PLGA NPs size distribution does not change after laser irradiation, although the NPs were more agglomerated after heating. Figure4 displays the release profile of unionized bupivacaine from uiBup-Pd PLGA NPs. Bupivacaine release present two stages: 1) an initial burst release of the entrapped drug probably close to the surface of the NPs (50% of bupivacaine is release after 5 h) and 2) a lag phase, when bupivacaine was slowly released (55% of the total bupivacaine load at 21 h), as observed with other PLGA-based particles for sustained drug delivery applications.[35b,55] The release profile was fitted to a Korsmeyer–Peppas diffusion model (Figure S5, Supporting Information) by calculating the transport exponent (n= 0.2) and transport constant (K= 0.1767 h−0.2). Y-axis intercept (0.41) is characteristic of the burst effect.[56] This burst initial release has been attributed to either the presence of nonencapsulated drug molecules on the surface of the NPs or to drug molecules that are located close to the external surface, but embedded in the polymer matrix.[57] The correlation coefficient (R2= 0.984) was rather high that indicates a good correlation with experimental data, and the transport exponent lower than 0.5 indicates a Fickian diffusion release from a nonswellable matrix.[58] The sustained release of bupivacaine without light irradiation is caused by the erosion of the PLGA NPs due to the hydrolytic degradation of the ester bonds present in its backbone.[59] As it is noted in Figure 4, laser irradiation caused a fast release of the 75% of the loaded drug (≈200 ppm) after 5 heating-cooling cycles. This can be attributed to the destabilization of PLGA because its glass transition temperature (Tg) is around 40 °C and it decreases over time.[60] Below the Tg, the polymer has limited mobility and low diffusion rates, and above it the polymer presents high water and drug transfer rates throughout the matrix.[61] So when the NPs are irradiated and reach 45 °C bupivacaine release increases due to a high diffusion rate being the thermal degradation of the polymer promoted. In addition, the interaction among drug-polymer evidenced by FTIR analysis (Figure S4, Supporting Information) diminishes with increasing temperature, in agreement with the previous literature.[62] Finally, Figure4 displays bupivacaine release of three different samples after 5 heating-cooling cycles activated by laser irradiation and at a release time of 0, 5, and 21 h, respectively. The drug released activated function was preserved in this time interval, concluding that uiBup-Pd PLGA NPs are stable light triggered drug delivery systems during the tested timeline. Furthermore, in order to corroborate the stability of the nanoparticles in a simulated biological medium, we have evaluated in vitro drug release in PBS supplemented with 10% fetal bovine serum (FBS). In addition, we have evaluated the long-term stability of the NPs (7 days) using SEM and TEM (Figure S6, Supporting Information). Results show a sustained release of bupivacaine during 24 h, which indicates that nanoparticles are stable without premature degradation. According to SEM and TEM images, morphology and size of the nanoparticles do not change over time. 2.5. In Vitro Photothermal Effects: 2D and 3D Cells Models Figure S7 (Supporting Information) shows the in vitro cytotoxic effects of Pd-PLGA NPs on B16F1 (mouse melanoma cells), THP-1 (macrophages), human dermal fibroblasts, mouse mesenchymal stemcells (mMSCs) and human epidermal keratinocytes (HaCat) cultures. The treatment of B16F1 and macrophages cells with the NPs did not exert cytotoxic effects at the concentration range assayed (0.02–2 mg mL−1) displaying viability percentages above 70%, which is considered as noncytotoxic according to the ISO 10993-5.[63] Moreover, macrophages did not show any effect after treatment for 24 h as their viability was the same as the one obtained for the nontreated samples (control samples). However, the NPs displayed cytotoxic effects at the higher concentrations assayed (1 and 2 mg mL−1) on HaCat, Figure 4. Bupivacaine release from uiBup-Pd PLGA NPs at 37°C(blue profile) and after 5 cycles of heating-cooling from 37 to 45°Cby laser irradiation (808nm, 1.8 Wcm−2, 2.4mgmL−1 of NPs) (red points) of 3 independent samples. Percentages are displayed as mean ± SD (N= 3). Adv. Funct. Mater. 2022, 32, 2106932 www.afm-journal.dewww.advancedsciencenews.com 2106932 (9 of 14) © 2021 The Authors. Advanced Functional Materials published by Wiley-VCH GmbH fibroblasts, and mMSCs cells, resulting in viability percentages around 40–60%. It should be noticed that the potential interference of the Pd-PLGA NPs with the fluorescence emission reading at the highest concentration tested was also evaluated and discarded. Previous studies have also shown the cytotoxic effects of PLGA NPs loading Pd complexes (1–50×10−6m) in human ovarian carcinoma cell (OVCAR3) cultures after 48 h of treatment.[64] Their results showed the low cytotoxicity of Pd complexes displaying viability percentages higher than 70%. However, the nanoparticulate system exerted significant cytotoxic effects resulting in viability percentages around 20–50% which are very different from our results as at the highest concentration assayed (2 mg mL−1) the Pd content was around 11.3 × 10−6 m showing viability percentages higher than 80%. Surprisingly, the effects in their assays were not dependent on the concentration. In fact, viability percentages were higher when the concentration was increased. These authors hypothesize that this effect may be attributed to the precipitation of Pd complexes due to their low solubility in water, while their loading into PLGA NPs favoured their slow release and their interaction with cell membranes. In addition, in their study Pd is present as a complex in its ionic form but in our case, Pd is present in its elemental (nonvalent form). On the other hand, PLGA-PEG NPs were loaded with Pd and doxorubicin, and the resulting nanoparticulate system was assayed for 72 h in cancer cells (HT1080).[65] Their results demonstrated a moderate cytotoxicity with a reduction in viability of 50% when adding a concentration of 35×10−6m to the cultures, while our assays were performed at lower concentrations (≤ 11.3×10−6m Pd) and for 24 h though achieving higher cell viability percentages. Photothermal effects derived from Pd-PLGA NPs treatment of melanoma cells (B16F1) and macrophages were evaluated by fluorescence microscopy through the staining of live cells with calcein (green) and dead cells with ethidium bromide (red). B16F1 cells (Figure5a–d) and macrophages (Figure S8, Supporting Information) photothermal damage was clearly observed in the irradiated area of each treated sample only after 5 min of irradiation. Non internalized NPs were washed out before irradiating the cultures. The treated but not irradiated samples (Figure5a–c; and Figure S8a–c, Supporting Information) exerted high cell viability showing mostly cells stained in green (live cells), which is in accordance with the results obtained in the cytotoxicity assays described above (Figure S6, Supporting Information). However, when samples were irradiated, the central area of the wells showed a red spot (Figure5b–d; and Figure S8b–d, Supporting Information) due to the red staining of dead cells mediated by ethidium bromide. This effect seems to be more evident when the concentration was increased. Figure S8b–d (Supporting Information) shows irradiated macrophages treated with 0.5 and 1mgmL−1 of Pd-PLGA NPs, respectively. The red spot is slightly wider in Figure S8d (Supporting Information) (1mgmL−1) and cell mortality is higher in the surrounding area compared to Figure S8b (Supporting Information) (0.5 mg mL−1) as red stained cells show. These results point to a dose dependent effect of the treatment with Pd-PLGA NPs after irradiation. Furthermore, melanoma cells (Figure 5a–d) displayed low viability after NP treatment and NIR irradiation as it is shown in Figure 5b–d. Indeed, a red spot is not observed in these samples due to the high mortality, which may involve the complete loss of cell adherence and thus, their elimination when cells were washed prior to microscopy visualization. These data are in accordance with the cytotoxicity assays explained above as melanoma cells were more sensitive to NP treatment compared to macrophages, which images (Figure S8b–d, Supporting Information) displayed the red spot in the irradiated area while not reducing considerably cell density. The dose dependent effect is also observed in melanoma samples treated with Pd-PLGA NPs and NIR irradiated as clearly demonstrates the lowest cell density at the highest concentration assayed (1mgmL−1; Figure5d). It is important to point out that the thermal effect did not macroscopically affect the whole well as the temperature increase measured was only 2 °C; therefore, the photothermal effect was confined on the irradiating spot area due to the coherent character of the laser beam. Finally, the photothermal effects in B16F1 cells were evaluated by flow cytometry after treatment with Pd-PLGA NPs and NIR irradiation (Figure S9, Supporting Information). We observed that under subcytotoxic doses (1mgmL−1) no statistically significant changes on cell cycle were observed when treating the cells with just the NPs, just the NIR light (at the required irradiances to generate optical hyperthermia) or with the combination of both nanoparticles and NIR light. No changes in apoptosis (early, late apoptosis, and necrosis) compared to untreated controls were observed either. Finally, no caspase 3 activation was detected by flow cytometry (data not shown). All in all, those results highlight the benign character of our NPs on the nontreated cells. After performing optical hyperthermia with those NPs on the metastatic melanoma cell line, all dead cells affected by the transduced heat were detached from the wells (being adherent cells when alive) and those dead cells were removed in the washing followed during the trypsin treatment used prior cell cytometry analysis. Some of the unaffected cells that remained adhered on the wells (those far away from the irradiating light spot) were the ones collected and evaluated by flow cytometry. We observed no changes in cell cycle of apoptosis induction or caspase 3 activation highlighting the lack of collateral effects and the benefits of the spatiotemporal control of the optical hyperthermia here described. After validating the photothermal activity of Pd-PLGA NPs in B16F1 and in macrophages as 2D cultures, the same assay was performed in 3D cell spheroids. Spheroids can mimic the environment and the interactions between cells of tumors.[66] Moreover, spheroids have been widely accepted as models of study for drug delivery systems[67] or photothermal therapy.[68] In this work, B16F1 spheroids were used to evaluate the in vitro efficiency of Pd-PLGA NPs combined with irradiation in photothermal therapy. In agreement with the results obtained in the 2D model, the Pd-PLGA NPs treatment without irradiation did not produce any cytotoxic effect on the spheroids (Figure 5f), showing mostly green stained cells (live cells), as the nontreated and nonirradiated control spheroids (Figure5e). Figure5g,h shows treated and irradiated samples with 0.5 and 1 mg mL−1 of Pd-PLGA NPs, respectively. Treated and irradiated spheroids displayed high cell mortality showing mostly cells stained in red (dead cells). In accordance to the 2D assay, the photothermal effect was dependent on the NPs concentration exerting higher cell mortality after treatment with Adv. Funct. Mater. 2022, 32, 2106932