pharmaceutics Article MRI/Photoluminescence Dual-Modal Imaging Magnetic PLGA Nanocapsules for Theranostics Yajie Zhang 1 , Miguel García-Gabilondo 2 , Anna Rosell 2, * and Anna Roig 1, * 1 Institut de Ciència de Materials de Barcelona (ICMAB-CSIC), Campus UAB, 08193 Bellaterra, Catalonia, Spain; yzhang@icmab.es 2 Neurovascular Research Laboratory, Vall d’Hebron Institut de Recerca, Universitat Autònoma de Barcelona, 08035 Barcelona, Catalonia, Spain; miguel.garcia@vhir.org * Correspondence: anna.rosell@vhir.org (A.R.); roig@icmab.es (A.R.)  Received: 30 October 2019; Accepted: 17 December 2019; Published: 21 December 2019  Abstract: Developing multifunctional and biocompatible drug delivery nanoplatforms that integrate high drug loads and multiple imaging modalities avoiding cross-interferences is extremely challenging. Here we report on the successful chemical reaction of the high quantum yield biodegradable and photoluminescent polyester (BPLP) with the poly(lactic-co-glycolic acid) (PLGA) polymer to fabricate biocompatible photoluminescent nanocapsules (NCs). Furthermore, we transform the PLGA-BPLP NCs into a magnetic resonance (MR)/photoluminescence dual-modal imaging theranostic platform by incorporating superparamagnetic iron oxide nanoparticles (SPIONs) into the polymeric shell. In vitro phantoms confirmed the excellent MRI-r2 relaxivity values of the NCs whilst the cellular uptake of these NCs was clearly observed by fluorescence optical imaging. Besides, the NCs (mean size ~270 nm) were loaded with ~1 wt% of a model protein (BSA) and their PEGylation provided a more hydrophilic surface. The NCs show biocompatibility in vitro, as hCMEC/D3 endothelial cells viability was not affected for particle concentration up to 500 µg/mL. Interestingly, NCs decorated with SPIONs can be exploited for magnetic guiding and retention. Keywords: PLGA nanocapsules; magnetic resonance imaging; photoluminescence; drug delivery systems; magnetic targeting; multimodal imaging; theranostics 1. Introduction Nanomedicine, which refers to the application of nanotechnology in medicine, offers valuable new tools for the diagnosis and treatment of many diseases. Nanoparticles are increasingly important by assisting to expedite the development of contrast agents, therapeutics, drug delivery vehicles, and theranostics in the context of nanomedicine [1]. Polymer-based nanoparticles are frequently proposed as drug carriers due to their biocompatibility and biodegradability, as well as the possibility of customizing their physicochemical properties for a specific drug or delivery route [2–4]. Among them, poly(lactic-co-glycolic acid) PLGA nanoparticles have gathered particular attention, since they encompass a number of interesting features: (i) FDA and European Medicine Agency approval in drug delivery systems for parenteral administration; (ii) well described formulations and methods of production adapted to various drugs i.e., hydrophilic or hydrophobic, small molecules, or macromolecules; (iii) protects the loaded drugs from degradation and possibility of sustained release; and, (iv) easy to modify to include targeted delivery or to provide tuned performance in a specific biological environment [5,6]. Theranostics is an emerging field that combines diagnostics and therapeutics into multifunction nanoparticle systems [7]. Drugs and nanocarriers in vitro/in vivo fate can be monitored while using noninvasive imaging techniques, such as magnetic resonance imaging (MRI) and fluorescent imaging, Pharmaceutics 2020, 12, 16; doi:10.3390/pharmaceutics12010016 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, x 2 of 16 Pharmaceutics 2020, 12, 16 2 of 16 noninvasive imaging techniques, such as magnetic resonance imaging (MRI) and fluorescent imaging, in order to optimize the route of delivery, biodistribution, and drug accumulation, among in order other to optimize factors the route [4,8,9]. PLGA of delivery, nanoparticles canbiodistribution, and drug be used in diagnostic andaccumulation, amongby therapeutic imaging other the factors addition[4,8,9]. of the PLGA nanoparticles imaging moieties can be used during the in diagnostic particle and therapeutic synthesis. imaging To date, large by the efforts addition have been of the imaging devoted moieties during to conjugating PLGA the withparticle synthesis. quantum semiconducting To date, large dots efforts have been and organic dyesdevoted to createto conjugating photoluminescent PLGA withPLGA semiconducting quantum nanocarriers [9–11]. dots andconventional However, organic dyes to create photoluminescent approaches for physically PLGA blendingnanocarriers imaging [9–11]. probesHowever, within conventional the nanoparticlesapproaches can for leadphysically blending conclusions to incomplete imaging probes or within the nanoparticles misinterpretations on thecannanoparticles’ lead to incomplete conclusionsand biodistribution or misinterpretations on the the fate [12,13]. Moreover, nanoparticles’ inevitable biodistribution photobleaching and and fate low[12,13]. Moreover, dye-to-polymer the inevitable labeling ratios ofphotobleaching organic dyes and andthe lowinnate dye-to-polymer toxicity of labeling quantumratiosdots of organictheir prevent dyespractical and the innate use intoxicity of quantum vivo [14,15]. dots prevent Recently, a series their practical use of biodegradable in vivo [14,15]. Recently, photoluminescent a series of(BPLPs) (PL) polyesters biodegradable photoluminescent could derived (PL) polyesters (BPLPs) inherently photoluminescent PLGA-BPLPcould derived copolymersinherently photoluminescent with excellent PLGA-BPLP biocompatibility, copolymers tunable with excellent luminescence biocompatibility, and degradation rates, and tunable good luminescence and degradation thermal and mechanical rates, and properties, thusgood thermalthe expanding and mechanicalapplications biomedical properties, thus of PLGAexpanding the to highly biomedical desired optical applications of PLGA to highly desired optical imaging [16,17]. imaging [16,17]. Here, we we present present PLGA PLGAnanocapsules nanocapsules(NCs) (NCs)asas a dual-modal a dual-modal imaging imaging theranostic theranostic platform platform for magnetic for magnetic targeting targetingprotein protein delivery delivery(Figure (Figure 1).1). WeWe report reportononthe theprotocol protocolfor for the the fabrication fabrication of magnetic PLGA-BPLP NCs with intrinsic photoluminescence photoluminescence and MRI capacity being endowed by the incorporation of superparamagnetic iron oxide nanoparticles (SPIONs). Moreover, Moreover, the NCs are functionalized with with poly(ethylene poly(ethyleneglycol) glycol)(PEG), (PEG),providing providinga ahydrophilic hydrophilic surface surfacethat could that could result in result anan in enhanced enhanced stealth effect. stealth effect.Unlike Unlikesolid solidPLGA PLGAnanoparticles nanoparticles[4,18,19], [4,18,19],in inour our NCs, NCs, the functional functional moieties are moieties areincorporated incorporatedinto intothethe polymeric polymeric shell shell matrix matrix to minimize to minimize interferences interferences with with the the cargo cargo being being placed placed inside theinside NC;thethisNC; this is especially is especially important important for payloads, for delicate delicate payloads, such as enzymes, such as proteins, proteins, enzymes, or or microRNAs. microRNAs. In thisbovine In this study, study, bovine serum albumin serum albumin (BSA) (BSA) has beenhas used been used as a model as a model protein protein for for evaluating evaluating the the protein protein loading loading capability capability andand release release kinetics kinetics of proposed of the the proposed nanocarrier. nanocarrier. Figure 1. 1.Schematic Figure illustration Schematic of poly(lactic-co-glycolic illustration acid)-biocompatible of poly(lactic-co-glycolic photoluminescent acid)-biocompatible polymer nanocapsule polymer photoluminescent (PLGA-BPLP NC) as a dual-modal nanocapsule imaging (PLGA-BPLP theranostic NC) platform. as a dual-modal imaging theranostic platform. 2. Experimental Section All of the reagents 2. Experimental Sectionwere purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless specified otherwise. All of the reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless specified 2.1. Synthesis otherwise. Copolymer of PLGA-BPLP The PLGA-BPLP copolymer was synthesized by a slight modification of Yang’s method [16,17]. 2.1. Synthesis of PLGA-BPLP Copolymer Step one involves the synthesis of a hydroxyl terminated BPLP pre-polymer. Briefly, citric acid, The PLGA-BPLP 1,8-octanediol, copolymer and l-cysteine was with thesynthesized molar ratioby ofa1:1:0.2 slight (5.76 modification g, 4.38 g,of0.72 Yang’s method g) were added[16,17]. into a flask of 50 mL with a stirring bar. Under a constant flow of argon, the reactants were melted1,8- Step one involves the synthesis of a hydroxyl terminated BPLP pre-polymer. Briefly, citric acid, by octanediol, heating and◦ C to 140 L-cysteine withsolution until a clear the molar ratio of formed. 1:1:0.2 The (5.76was solution g, 4.38 g, 0.72tog)react allowed wereatadded into a flask this temperature of 50 for 80mL with min. and a stirring bar. Under then stopped beforea the constant flow stirring barofstopped argon, the reactants stirring were melted completely in theby heating to increasingly 140 °C until viscous a clear solution by solution adding 25formed. mL of The solutiontowas 1,4-dioxane allowed dilute to react at the produced this temperature prepolymer. for 80 min. The product was and thenbystopped purified dropwise before the stirring precipitation from barthe stopped stirring 1,4-dioxane completely solution in waterin to theremove increasingly viscous the unreacted solution by The monomers. adding final25 mLproduct BPLP of 1,4-dioxane to dilute was collected the producedand by centrifugation prepolymer. The product was then lyophilized. Pharmaceutics 2020, 12, 16 3 of 16 In the second step, the PLGA-BPLP copolymer was synthesized while using the BPLP as a macroinitiator to react with l-lactide and glycolide via a ring-opening polymerization that was catalyzed by Tin (II) 2-ethylhexanoate (Sn(OCt)2 ). Briefly, l-lactide, glycolide and BPLP with the molar ratios of (75:25):1 or (50:50):1 were added into a reaction tube. Subsequently, Sn (OCt)2 (0.1 wt% of l-lactide and glycolide mixture) was added as a solution in dichloromethane which was then evaporated in vacuum. The tube was flushed with argon and capped and subsequently immersed in a 160 ◦ C oil bath for 48 h. The obtained product was dissolved in chloroform and then purified by dropwise precipitation into pure ethanol to remove unreacted raw materials. Finally, the PLGA-BPLP copolymer was recovered by centrifugation and then dried in vacuum at room temperature. All of the PLGA-BPLP mentioned in the text were with molar ratios of (75:25):1, unless specified otherwise. 2.2. Synthesis of Oleic Acid Coated SPIONs The SPIONs were synthesized by microwave assisted thermal decomposition in a microwave synthesizer (Discover SP, CEM Corporation, Matthews, NC, USA), and then coated by oleic acid (OA) [20]. The process to obtain homogeneous OA-SPIONs with an average diameter of 9 nm was as follows: 3.5 mmol of iron precursor Fe (acac)3 was dissolved in 4.5 mL of benzyl alcohol in a microwave reaction glass tube. Microwave irradiation was initiated at 60 ◦ C for 5 min. to fully dissolve the precursor and, subsequently, the irradiation was kept at 210 ◦ C for 30 min., and the reaction was then stopped and cooled down to room temperature. 4 mL of oleic acid in toluene (0.8 mmol/mL) was added immediately into the as-synthesized SPIONs dispersion followed by incubation under ultrasound for 1 h. Subsequently, the obtained OA-SPIONs were separated by centrifugation in five-fold of acetone. The pellet was redispersed in 4 mL of toluene in a glass vial and a magnet was attached on the wall for 5 s, the un-adsorbed suspension containing SPIONs with smaller size was discarded and the adsorbed pellet was redispersed in 6 mL of dichloromethane (DCM), followed by centrifugation at 4000 rpm for 5 min. to sediment the unstable big particles. Finally, the stable OA-SPIONs (9 nm) dispersion in DCM was centrifugated in five-fold of acetone and the pellet product was dried under vacuum and redispersed in DCM at the concentration required for use. 2.3. Fabrication of Functional PLGA-BPLP NCs and Encapsulation of BSA Functional PLGA NCs encapsulating BSA were prepared by a double emulsion solvent evaporation method. Briefly, 50 µL of inner aqueous phase (W1) containing BSA (30 mg/mL) was emulsified in 500 µL of DCM organic phase (O) that was composed of 50 mg of different proportions of PLGA (RG502H, Mn 12,000)/PLGA-BPLP/PLGA-PEG (PLGA Mn 7000, PEG Mn 5000) and a certain amount of OA-SPIONs by sonication (VC505, Sonics & Materials Inc., Newtown, CT, USA) at 200 W for 28 s to form the first emulsion (W1/O). Afterwards, 2 mL of external aqueous phase (W2) with polyvinyl alcohol (PVA) (20 mg/mL) was added and the second emulsion (W1/O/W2) was formed by sonication for another 28 s. The temperature during the whole emulsion process was kept at 4 ◦ C by using an ice bath. The resulting double emulsion was poured into 50 mL of MilliQ water and mechanically stirred at RT for 2 h to allow for complete evaporation of the organic solvent DCM and the formation of NCs. Finally, the NCs were washed three times with MilliQ water and lyophilized in 6 mL of trehalose aqueous solution (2 mg/mL). The as-obtained powder was stored at 4 ◦ C with desiccant silica gel. The non-PEGylated and PEGylated NCs have been labelled, as follows: NC1.non PEGylated (90 wt% PLGA-BPLP + 10 wt% PLGA) and NC2.PEGylated (90 wt% PLGA-BPLP + 3 wt% PLGA + 7 wt% PLGA-PEG), both containing ~6 wt% of SPIONs, as gathered in Table 1. Pharmaceutics 2020, 12, 16 4 of 16 Table 1. Summary of the two main formulations for the NCs. Oleic acid coated-SPIONs and PLGA-BPLP and PLGA-PEG were mixed in the organic phase during the double miniemulsion process. Shell Polymers (wt%) SPIONs Ms Size (DLS) BSA NCs Type BSA Loading (eum/g) Loading PLGA PLGA-BPLP * PLGA-PEG d. nm PdI EE% (wt%) (wt%) NC1.non-PEGylated 10 90 / 5.7 4.0 272 0.11 0.96 38.5 NC2.PEGylated 3 90 7 6.0 4.2 265 0.05 1.01 41.3 * PLGA-BPLP from initial molar ratios (LA:GA):BPLP = (75:25):1. 2.4. Physicochemical Characterization of PLGA-BPLP, SPIONs, and NCs 2.4.1. Absorption Spectroscopies of the Polymers Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) characterization of the polymers was performed on a Bruker Vertex 70 FTIR spectrometer with a Pike Miracle Single-Bounce diamond crystal plate accessory at room temperature. The FTIR spectra were recorded over a wavelength range of 4000–500 cm−1 with a resolution of 4 cm−1 . UV-Vis absorbance of the fluorescent polymers was recorded on a Varian Cary-5000 UV-Vis spectrophotometer while using a quartz cuvette with an optical path of 1 cm. 2.4.2. Size Distribution and Disperse Stability of the NCs Dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Panalytical, Madrid, Spain) measurement of the hydrodynamic diameter and size distribution of NCs by intensity was performed by redispersing 0.5 mg of lyophilized NCs powder into 1 mL of MilliQ water. Turbiscan (Turbiscan Lab, Formulaction, Toulouse, France) is used to detect the destabilization of the NCs suspension. 15 mL of 2 mg/mL NCs in the Turbiscan cell were scanned at all of the heights of the suspension with a time interval of 2 min. during 24 h. The back scattering signals at different heights of the cell were recorded and the delta of back scattering intensity was calculated by subtracting the reference time 0 s. The bottom part was defined as 1/5 of the liquid level. Nanosight (NS300, Malvern Panalytical, Madrid, Spain) is used to measure the averaged size and concentration of the NCs water suspension. 0.2 mg/mL of NCs with 50 times dilution was pumped into the cell and the data were acquired and analyzed through the Nanoparticle Tracking Analysis (NTA) software along with the instrument. 2.4.3. Electron Microscopies Field emitting scanning electron microscope (SEM, FEI Quanta 200 FEG, Thermo Fisher Scientific, OR, USA) and transmission electron microscope (TEM, JEM-1210, JEOL Ltd., Tokyo, Japan) were used to study the morphologies of SPIONs and NCs. 0.5 mg of lyophilized powder was redispersed into 1 mL of MilliQ water and centrifuged at 4000 rpm for 10 min. for the SEM sample preparation of NCs. Subsequently, the supernatant was discarded to remove the trehalose (used for cryopreserving during lyophilization) and 1 mL of fresh water was added, the pellet of NCs was redispersed in water with ultrasound. Finally, 6 L of the slightly turbid suspension was deposited onto a small slice of silicon wafer stuck on the top of a carbon layer and dried at room temperature overnight. The sample was sputtered with Au-Pd (20 mA 2 min, Emitech K550, Quorum Technologies Ltd., East Sussex, UK). The TEM samples were prepared by placing and drying one drop of the corresponding NCs or SPIONs dispersion on a copper grid at room temperature. 2.4.4. Magnetometry Superconductive quantum interference device (SQUID, MPMS5XL, Quantum Design, San Diego, CA, USA) was used to measure the magnetization of NCs and SPIONs and calculate the SPIONs loading (wt%-SPIONs) of the magnetic NCs. Zero-field cooling and field cooling (ZFC-FC) measurement were used to determine the blocking temperature (TB ) of the SPIONs. A gelatin capsule filled with about Pharmaceutics 2020, 12, 16 5 of 16 7 mg of samples, together with some cotton wool, was inserted into the SQUID magnetometer sample holder and the hysteresis loop was measured from −50 kOe to 50 kOe. The saturation magnetization of the NCs (MS -NCs, emu/g) and of SPIONs (MS -SPIONs, emu/g) was used to calculate wt%-SPIONs, as follows: wt%-SPIONs = MS -NCs/MS -SPIONs × 100% 2.4.5. Fluorescence Properties Fluorescence spectra of the polymers and NCs were acquired on a spectrofluorometer (LS45, PerkinElmer Inc., Waltham, MA, USA). The excitation and emission slit widths were both set at 10 nm. The quantum yield of the polymers was measured by the Williams’ method [16]. Briefly, a series of BPLP/PLGA-BPLP solutions in the corresponding solvents were prepared with gradient concentrations. Maximal excitation wavelength was determined, which generated the highest emission intensity. The fluorescence spectra were collected for the series of solutions in the 10 mm fluorescence cuvette (Figure S1). The integrated fluorescence intensity, which is the area of the fluorescence spectrum, was calculated and then noted. Afterwards, the UV-Vis absorbance spectra were collected with the same solutions and the absorbance at the maximal excitation wavelength within the range of 0.01–0.1 Abs units was noted (Figure S1). The graphs of integrated fluorescence intensity vs. absorbance were plotted. The quantum yield was calculated according to the equation: Slopes ns 2 Φs = Φr ( )( ) Sloper nr where, Φ = quantum yield; Slope = slope of the straight line obtained from the plot of intensity vs. absorbance; n = refractive index of the solvent; s = subscript denotes the sample; and, r = subscript denotes the reference used. Here, anthracene (Φ = 27% in ethanol when excited at 366 nm) was used as the reference. The fluorescence intensities of different concentrations of NCs in water were quantified in a 96-well by a microplate reader (Spark, Tecan Group Ltd., Männedorf, Switzerland). The NCs were excited at the maximal excitation wavelength and the fluorescence signal was collected by area scan at the maximal emission wavelength. Photostability was measured by continuously illuminating the polymer or NCs in a fluorescent confocal microscope (Leica SP5, Leica Microsistemas S.L.U., Barcelona, Spain) while using UV diode 405 nm excitation at different laser power (10%, 20%, 100%). The fluorescence images were acquired at a time interval of 1 s for 10 min., changes of mean fluorescence intensity of six region of interests (ROIs) in 10 min. were calculated while using software Las AF. The real laser power (W) at different percentage during 10 min. was monitored using a laser power meter. 2.5. MRI Phantoms of the NCs In vitro agarose phantoms of NCs were prepared in Eppendorfs where a series of concentrations of NCs were vortexed and sonicated in agarose water solutions before the gel formed. The volume was kept at 1 mL with 0.63 wt% of agarose (Conda, Madrid, Spain). The corresponding iron doses (mmol/L) were calculated according to the wt% of SPIONs in each sample. T2 maps of the phantoms were acquired at 7 T in a 70/30 Bruker USR Biospec system (Bruker GmbH, Ettlingen, Germany), as follows: multi-slice multi-echo (MSME) sequence with echo time (TE) = 13 ms, repetition time (TR) = 4000 ms, field of view (FOV) = 5.5 × 11 mm, and three slices of 1 mm thickness. The quantitative T2 values were obtained from hand-drawn ROIs by using curve fitting in the Image Sequence Analysis (ISA) software along with the instrument. Pharmaceutics 2020, 12, 16 6 of 16 2.6. In Vitro Toxicity Evaluation of the NCs Two parallel methods assessed the toxicity of the NCs on human brain endothelial cells (hCMEC/D3): a viability assay based on WST-8 tetrazolium salt reduction (cell counting Kit-8, Dojindo) and direct cell counting. First, 104 viable cells per well were seeded on a 24-well plate pre-treated with collagen (rat tail type I, Corning) in 400 µL of endothelial growth medium (EGM2 from Lonza with 2% fetal bovine serum and half the amount of the growth factors that were included in the kit). After incubation for 72 h at 37 ◦ C with 5% of CO2 , the cells were at 80–90% confluence and medium was changed to endothelial basal medium (EBM2, Lonza) containing NCs at 25, 50, 100, 500, and 1000 µg/mL. After 48 h, the cells were washed and incubated with 10% WST-8 solution for two hours. Culture supernatants were centrifuged at 20,000 rpm for 5 min. to remove NCs detritus that could interfere with the dye absorption before determining the absorbance at 450 nm. Cells were trypsinized and resuspended in growth medium and diluted 1:1 in Trypan Blue in order to perform cell counting in a Neubauer chamber. Cell viability and count are expressed as the percentage of absorbance or number of cells as compared with the control (vehicle without NCs). ANOVA test and Dunnett’s multiple comparisons post-hoc test was performed vs. the control. 2.7. In Vitro Observation of NCs Cellular Uptake The hCMEC/D3 endothelial cells were seeded in cover-slips that were pre-treated with collagen (2 × 104 cells/well in 24 well plates) and 24 h later were exposed to 50 µg/mL of empty fluorescent NCs. After an additional 24 h of culture, the wells were washed with PBS, fixated with 4% paraformaldehyde, and mounted with Vectashield antifade mounting medium (Vector Laboratories) with propidium iodide for nucleic acid counterstaining. Additionally, some of the cells were stained with PKH26 lipophilic fluorescent dye for cell membrane labeling according to the manufacturer’s protocol. Images at 63× were obtained on a fluorescent confocal microscope (LSM 980 with Airyscan 2 detector, Zeiss, Oberkochen, Germany). 2.8. BSA Loading in NCs and Release Kinetics The albumin content of the NC was directly determined while using the CBQCA protein assay kit (Invitrogen™ ref. C6667), which determines the protein concentration based on the production of fluorescent products measurable at λex /λem = 450 nm/550 nm via non-covalent interaction between CBQCA and primary aliphatic amines of proteins. This highly sensitive fluorescence-based method showed compatibility with DMSO, SPIONs, detergents, and other substances that interfere with other commonly used protein determination methods. Lyophilized NCs encapsulating albumin as well as empty NCs as control were fully dissolved in DMSO at 100 mg/mL. The protein contents in the NCs lysates were measured and calculated based on the difference in fluorescence with the control and a calibration curve drawn with standard albumin solutions, the protein contents in the BSA solutions used for encapsulation were also measured. As listed in Table 1 for the two types of NCs, at least two replicate NCs batches of each were measured for the BSA loading and encapsulation efficiency (EE%) calculation. All of the measurements were performed in duplicate for each NCs batch. The experimental BSA loading in the NCs is expressed as µg of BSA per mg of NCs (µg/mg) or the wt% of the NCs, and the BSA EE% is calculated, as follows: Experimental BSA loading EE% BSA = × 100% Nominal BSA loading For the release studies, lyophilized NCs were resuspended in phosphate buffered saline (PBS) (pH 7.4, Sigma ref. D1408) at 10 mg/mL in low protein binding microcentrifuge tubes (Thermo Scientific© ref. 90410). NCs solutions were incubated at 37 ◦ C in a vertical rotator for different time measures to simulate the in vivo environment: right after the resuspension (time 0), and after three hours, six h, one day, and seven days of incubation. In all cases, an aliquot of 200 µL was frozen Pharmaceutics 2020, 12, 16 7 of 16 at −80 ◦ C until protein determination. Before the CBQCA assay, the aliquots were centrifuged at 15,000× g rcf to separate supernatant and pellet. The amount of protein release was directly calculated as total released protein and indirectly from the remaining protein in the pellet as indirect measure. For this purpose, the pellets were fully dissolved in DMSO and then compared with the intact NCs fully dissolved in DMSO (100 mg/mL) used as the 100% release set up. Four BSA-loaded NCs batches and one of H2 O-NCs batch as control were used and measures done in duplicate. The release profiles were expressed in terms of cumulative release and plotted vs. time. 3. Results and Discussion 3.1. Photoluminescent PLGA-BPLP Copolymer BPLPs are degradable oligomers synthesized from biocompatible monomers, including citric acid, aliphatic diols, and various amino acids via a convenient and cost-effective polycondensation reaction. BPLPs present some advantages over the traditional fluorescent organic dyes and quantum dots due to their cytocompatibility, minimal chronic inflammatory responses, controlled degradability, and excellent fluorescence properties [16]. Here, l-cysteine was selected and introduced into the polyester structure that was made of biocompatible monomers of citric acid and aliphatic 1,8-octanediol, since previously reported BPLP from this starting amino acid exhibited the highest quantum yield (62.3%) [16]. The fluorophore structure of BPLP was verified as a fused ring structure ((5-oxo-3,5-dihydro-thiazolopyridine-3,7-dicarboxylic acid, TPA) [21]. ATR-FTIR was also used to confirm the chemical structure of the as-synthesized BPLP (Figure 2A). Strong absorptions from the molecular backbone of the polyester were observed i.e., peaks at 1044 cm−1 , 1176 cm−1 , and 1716 cm−1 are attributed to the C=O stretch, C–O asymmetrical, and symmetrical stretches of the ester bond, respectively, the peaks at 2930 cm−1 and 2856 cm−1 are attributed to the C-H stretches of alkane from 1,8-octanediol and the band near 3467 cm−1 is from the −OH. NH bending of the secondary amide at 1527 cm−1 and −SH at 2575 cm−1 confirm that l-cysteine is chemically bound to the poly(diol citrate) chain. The shoulder band near 1635 cm−1 is attributed to the C=O stretching of the tertiary amide from the TPA ring. The average molecular weight (Mw ) of BPLP measured by matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF-MS) was 1044 g/mol (Figure S2). The BPLP oligomer served as a macroinitiator to react with l-lactide and glycolide via a ring-opening polymerization to produce PLGA-BPLP [17]. The as-synthesized PLGA-BPLP (75:25):1 with molar ratios equal to 75:25 for l-lactide to glycolide and equal to 1:100 for BPLP to total l-lactide and glycolide was reported to have desirable glass transition temperature (Tg, 32.5 ◦ C), mechanical properties, fluorescence properties, and degradation rate of the resulting product [17]. Moreover, we have proved here that the 75:25 formulations are more suitable for the fabrication of NCs by a double mini-emulsion method than the PLGA-BPLP (50:50):1 one, as shown in the following section. The obtained PLGA-BPLP copolymer (Figure 2B inset) exhibits the inherent photoluminescence from the BPLP. The similarities of bands and shapes of the IR spectra of the as-synthesized PLGA-BPLP and commercial PLGA (Figure 2B), for instance, bands at 1084 cm−1 , 1165cm−1 , and 1747 cm−1 from the ester bonds in PLGA indicate their similar chemical structure, given the fact that BPLP is a very small portion of the PLGA-BPLP copolymer. The fluorescence of the as-synthesized BPLP and PLGA-BPLP was evaluated and Figure 3A,B depict the excitation and emission spectra. The similar spectra further confirm the inherent photoluminescence of PLGA-BPLP from BPLP. Importantly, the fluorescence intensity of PLGA-BPLP only slightly decreased (10%) after 10 min. of continuous illumination under confocal microscope at 10% of laser power (0.40 ± 0.01 µW) (Figure 3C). When considering the laser power applied to observe stained cells is generally less than 10%, our photoluminescent polymer would exhibit good photostability at in vitro conditions. The calculated high quantum yields of BPLP (64%) and PLGA-BPLP (33%) from Figure 3D are consistent with the previously reported values [16,17]. The remarkable fluorescence properties that Pharmaceutics 2020, 12, 16 8 of 16 are shown here endow the PLGA-BPLP copolymer with high potential for the fabrication of functional Pharmaceutics 2020, 12, x 8 of 16 photoluminescent Pharmaceutics 2020, 12, NCs. x 8 of 16 Figure 2. Attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of the as-synthesized biocompatible Attenuated photoluminescent totalreflectance-Fourier reflectance-Fourier polymer transform (BPLP) infrared and PLGA-BPLP (ATR-FTIR) spectra Figure Figure 2.2. Attenuated total transform infrared (ATR-FTIR) spectra of the of the copolymer as-synthesized confirming biocompatibletheir chemical structures photoluminescent polymerand the (BPLP) successful and synthesis. PLGA-BPLP (A) BPLP; copolymer (B) confirming as-synthesized biocompatible photoluminescent polymer (BPLP) and PLGA-BPLP PLGA-BPLP their and commercial chemicalconfirming structures and PLGA synthesis. thechemical successful as reference. BPLP; Inset: (B) fluorescence ofand thecommercial PLGA-BPLP copolymer under a UV lamp. their structures(A) PLGA-BPLP and the successful synthesis. (A) BPLP;PLGA (B) as reference. Inset: fluorescence of the PLGA-BPLP under a UV lamp. PLGA-BPLP and commercial PLGA as reference. Inset: fluorescence of the PLGA-BPLP under a UV lamp. Figure Figure 3. (A,B) 3. (A,B) excitation excitation andand emission emission spectra spectra of the of the as-synthesized as-synthesized BPLPBPLP and and PLGA-BPLP. PLGA-BPLP. Insets: Insets: fluorescence of BPLP and PLGA-BPLP dispersed in solutions under a fluorescence of BPLP and PLGA-BPLP dispersed in solutions under a UV lamp; (C) photostability UV lamp; (C) photostability evaluation evaluation of PLGA-BPLP of PLGA-BPLP powder powder under confocal under confocal microscope microscope at different laser power, atfluorescence different laser 3. Figure intensitypower, (A,B)fluorescence excitation expressed as the andintensity expressed emission percentage vs.spectra as of the the value theatpercentage the initial vs. as-synthesized the time; value BPLP and, and (D)atPLGA-BPLP. the initial fluorescence time; fluorescence Insets: and, (D) fluorescence intensity-absorbance curves of BPLP andintensity-absorbance of BPLP, PLGA-BPLP, PLGA-BPLP and curves anthracene dispersed of as inused BPLP, PLGA-BPLP, a reference solutions under aused and(C) UVtolamp; calculate anthracene quantum yields.used as a reference used to calculate quantum yields. photostability evaluation of PLGA-BPLP powder under confocal microscope at different laser power, fluorescence intensity expressed as the percentage vs. the value at the initial time; and, (D) fluorescence intensity-absorbance curves of BPLP, PLGA-BPLP, and anthracene used as a reference used to calculate quantum yields. Pharmaceutics 2020, 12, 16 9 of 16 3.2. Fabrication of PLGA-BPLP NCs Combining Other Functional Moieties PLGA-based NCs with BSA encapsulated were prepared by a double emulsion solvent evaporation method with slight modifications from our previously reported method [22]. Functional moieties, such as PLGA-BPLP for the fluorescence imaging, PLGA-PEG to increase hydrophilicity and sealthness, and SPIONs for magnetic targeting and MRI (Figure 4A,B) can be incorporated to the organic phase during the NCs fabrication process (see Table 1). The first important remark is that NC morphology was not affected, even when using 100 wt% of PLGA-BPLP (75:25):1 (Figure S3A). A formulation with 90 wt% of PLGA-BPLP (75:25):1 was selected to allow for NC PEGylation by the addition of 7 wt% of PLGA-PEG and for the benefit of higher fluorescence intensity (Figure S4). Note that 7 wt% of PLGA-PEG (3 wt% PEG, Figure S3B) (NC2) was found to be the maximum amount that can be mixed in the organic phase during the NCs fabrication process, due to the amphiphilic property of PLGA-PEG. For a larger wt% of PLGA-PEG, the morphology and size of NCs were not maintained. As shown in the SEM image (Figure 4C), non-PEGylated and PEGylated NCs, as listed in Table 1, both contain ~6 wt% of SPIONs and depict homogeneous spherical morphologies and sizes (d.nm ~270) being similar to other reported PLGA systems that are suitable for intravenous administration [23]. The upper inset in Figure 4C shows a representative broken NC, exposing the hollow core where the protein drug is loaded. We have also found that the PLGA-BPLP (50:50):1 polymer from using initial molar ratio of LA:GA = 50:50 and BPLP:(LA + GA) = 1:100 was not as suitable for the fabrication of NCs. NCs with homogeneous morphology and narrow size distribution were attained only up to a maximum of 30 wt% of modified PLGA (Figure S3C) and without the possibility of further adding PLGA-PEG when using PLGA-BPLP (50:50):1 (Figure S3D). These results are in accordance with the higher glass transition temperature and better mechanical properties of PLGA-BPLP (75:25):1 over those of PLGA-BPLP (50:50):1 [17]. As expected, the NCs with a higher fraction of PLGA-BPLP (75:25):1 (90 wt%) show higher fluorescence intensity than the ones that were obtained with PLGA-BPLP (50:50):1 (30 wt%) at the same concentration (Figure S4). Regarding magnetic loading, up to 6 wt% of SPIONs could be loaded without affecting the NCs morphology and yielding a saturation magnetization (MS ) value of around 4 emu/g NCs (Figure 4D). In addition, the lower blocking temperature (TB , 33 K) of SPIONs in the NCs (Figure 4D inset) than the SPIONs of dry powder (55 K, Figure 4B inset) further demonstrates that the SPIONs are well dispersed in the polymer matrix. Note that a high magnetic loading is desirable for the magnetic retention of NCs. This is illustrated in the inset images of Figure 4D, where the darker-coloured water suspension of NCs with 6 wt% of SPIONs were adsorbed faster to the tube wall on the magnet side than the NCs with 1 wt% SPIONs loading at the same concentration, promisingly benefiting the magnetically targeted drug delivery as compared to the previously reported results [4,24]. Note that the superparamagnetic behaviour of NCs at room temperature (lack of coercivity, Figure S5) ensures no magnetic interactions among NCs in the absence of an external magnetic field, minimizing the risk of embolization during i.v. administration. Pharmaceutics 2020, 12, 16 10 of 16 Pharmaceutics 2020, 12, x 10 of 16 Figure4.4. (A) Figure (A) Representative Representativetransmission transmission electron microscope electron microscope(TEM) image (TEM) of theofoleic image acid (OA) the oleic acid coated SPIONsSPIONs (OA) coated with the with upperthe insetupper showing theshowing inset selected area the electron selecteddiffraction (SAED) area electron pattern of diffraction maghemite and lower (SAED) pattern inset a size and of maghemite distribution histogram lower inset a sizeof the particleshistogram distribution in the image; of (B) thehysteresis particles loop (5 K) and ZFC-FC (inset) for the OA-superparamagnetic iron oxide nanoparticles in the image; (B) hysteresis loop (5 K) and ZFC-FC (inset) for the OA-superparamagnetic (OA-SPIONs), inserted picture shows the stable OA-SPIONs in dichloromethane attracted by an external magnet; iron oxide nanoparticles (OA-SPIONs), inserted picture shows the stable OA-SPIONs in (C) representative SEM image of lyophilized NCs with the upper inset showing the hollow core of a dichloromethane attracted by an external magnet; (C) representative SEM image of nanocapsule and lower inset a TEM image of three NCs with the SPIONs visible as black spots well lyophilized NCs with the upper inset showing the hollow core of a nanocapsule and lower distributed in the polymer matrix; (D) hysteresis loop (5 K) and ZFC-FC (inset) for the lyophilized inset a TEM image of three NCs with the SPIONs visible as black spots well distributed in nanocapsule batch NC1, inserted pictures show the water suspension of these NCs (2 mg/mL) where the polymer matrix; (D) hysteresis loop (5 K) and ZFC-FC (inset) for the lyophilized the 6 wt% loading of SPIONs were adsorbed faster to the tube wall on the magnet (diameter 8mm, nanocapsule batch NC1, inserted pictures show the water suspension of these NCs (2 surface field ~0.4 T) side than that of 1 wt% loading. mg/mL) where the 6 wt% loading of SPIONs were adsorbed faster to the tube wall on the magnet It is well(diameter reported8mm, that surface field PEGylation the surface ~0.4 T) side than that of 1 wt% of engineered loading. nanoparticles provides them with stealth character increasing blood circulation time since nanoparticles are less visible to the It is well reported reticulo-endothelial that [6]. system the surface PEGylation We evaluated of engineered the flocculation nanoparticles regime of the NCsprovides them with with a Turbiscan to stealth character increasing blood circulation time since nanoparticles are less visible confirm successful surface modification with PEG, since the long hydrophilic PEG chains (Mn 5000) on to the reticulo- endothelial the surface ofsystem the NCs[6].are We evaluated expected the flocculation to increase regime the stability ofNCs of the the NCs withsuspension in water a Turbiscan andtodecrease confirm successful the surface modification sedimentation with PEG, rate. Sedimentation since of the thesuspension NCs long hydrophilic PEG chains was monitored for(Mn 24 h.5000) Figureon the 5A surface of the NCs are expected to increase the stability of the NCs in water suspension shows that, as NCs sedimentation progresses, the back scattering signal of the bottom part of the and decrease the sedimentation suspension increasesrate. fromSedimentation an increasingly of the NCsconcentration higher suspension was monitored of NCs, for signal while the 24 h. Figure 5A of the top shows part that, as NCs decreases. sedimentationrates The sedimentation progresses, the back scattering of non-PEGylated signal NCs and PEGylated of the bottom were part ofwith compared the suspension increases from an increasingly higher concentration of NCs, while or without a physiological concentration of BSA (0.5 mM) in Figure 5B, as expected the bottom back the signal of the top part decreases. scattering signalThe sedimentation of the PEGylated NCs ratesmedia of non-PEGylated increases at aand PEGylated slower rate thanNCsthe were compared ones non-PEGylated with or without a physiological concentration of BSA (0.5 mM) in Figure 5B, as expected the bottom back scattering signal of the PEGylated NCs media increases at a slower rate than the non-PEGylated ones both with and without BSA, which demonstrates a better dispersibility of the NCs due to the surface Pharmaceutics 2020, 12, 16 11 of 16 Pharmaceutics 2020, 12, x 11 of 16 both with and without BSA, which demonstrates a better dispersibility of the NCs due to the surface hydrophilic hydrophilicPEGPEGchains. chains.Note Note that thethe that sedimentation sedimentation rates of non-PEGylated rates of non-PEGylated and PEGylated and PEGylatedNCs both NCs slow both down with the slow down withphysiological concentration the physiological of BSA concentration ofprobably due todue BSA probably thetointeraction of NCs the interaction ofwith NCs the dense BSA solution. Additionally, in Figure 5A, the back scattering signal of the with the dense BSA solution. Additionally, in Figure 5A, the back scattering signal of the middle part middle part did not vary with time, which means that the NCs were monodispersed at the did not vary with time, which means that the NCs were monodispersed at the physiological physiological concentration of BSA and flocculation concentration of BSA and or coalescence flocculationdid not occur during or coalescence did not theoccur 24 h period. during the This24ishinperiod. consistent Thiswith is in the DLS size distribution results that are shown in Figure 5C, both non-PEGylated consistent with the DLS size distribution results that are shown in Figure 5C, both non-PEGylated and PEGylated NCs remained monodisperse and PEGylated at the physiological NCs remained monodisperse concentration of BSA, which at the physiological is of greatofadvantage concentration BSA, whichforisthe of i.v. administration great advantage for and theini.v. vivo blood circulation. administration and inNanosight vivo bloodwas also usedNanosight circulation. as an additional technique was also used as for an the determination additional of size technique anddetermination for the the concentration of the of size and NCs the(Figure 5D). Theofresults concentration the NCs show a similar (Figure 5D). size The distribution results showasaobtained similar sizeby DLS. From the distribution asnumber obtained concentration by DLS. From of the the NCs, number we concentration can determine of a mean mass of 1.06 × 10 −11 mg/NC. the NCs, we can determine a mean mass of 1.06 × 10 mg/NC. −11 Figure5.5.(A) Figure Back (A) Backscattering intensity scattering change intensity of the NC2 change of the PBS NC2suspension (shown inset) PBS suspension containing (shown inset) 0.5 mM of BSA at different height of the vial along 24 h measured by Turbiscan; containing 0.5 mM of BSA at different height of the vial along 24 h measured by Turbiscan; (B) quantified back scattering intensity (B) quantified backchange of the intensity scattering bottom part of the of change vial along the 24 h part bottom for NCs suspension of the in different vial along 24 h for media measured by Turbiscan; (C) dynamic light scattering (DLS) size distributions NCs suspension in different media measured by Turbiscan; (C) dynamic light scattering of NC1 and NC2 PBS suspensions with 0.5 mM of BSA along 24 h; (D) quantitative number (DLS) size distributions of NC1 and NC2 PBS suspensions with 0.5 mM of BSA along 24 concentration and size h; distribution of the nanocapsules measured by Nanosight (n = 3, mean ± SD with (D) quantitative number concentration and size distribution of the nanocapsules measured error bar). by Nanosight 3.3. Imaging (n = 3,ofmean Performance ± SD with the Magnetic error bar). NCs Photoluminescent The fluorescence of the NCs was evaluated, and Figure 6A depicts the excitation and emission 3.3. Imaging Performance of the Magnetic Photoluminescent NCs spectra. The spectra are similar to those of the PLGA-BPLP polymer shown in Figure 3B. Importantly, The fluorescence the incorporation of theinNCs of SPIONs was evaluated, the polymer matrix and doesFigure 6A depicts not quench the excitation the fluorescence andNote of NCs. emission that aspectra. The spectra are small displacement similar of the to those emission peakofwavelength the PLGA-BPLP polymerfor was observed shown in Figure aqueous 3B. Importantly, dispersed fluorescent the incorporation NCs when compared of to SPIONs in thepeak the emission polymer of thematrix polymerdoes in anot quench the chloroform fluorescence solution of NCs. (Figure 3B), whichNote we that a small ascribe to thedisplacement of the emission different interaction peak wavelength of the fluorescent probe with was theobserved for aqueous two solvents. dispersed The fluorescence fluorescentof intensities NCs NCs when show compared a lineartodependency the emissionon peak theofNCsthe polymer in a chloroform concentration within a solution range of(Figure 0.1 to 3B),mg/mL; 1.0 which we ascribeconcentrations at higher to the differenttheinteraction of the fluorescence fluorescent shows a trendprobe withsaturation towards the two solvents. The (Figure 6B). fluorescence intensities of NCs show a linear dependency on the NCs concentration within a range of 0.1 to 1.0 mg/mL; at higher concentrations the fluorescence shows a trend towards saturation (Figure 6B). NCs can be clearly imaged with a fluorescence confocal microscope and they show a very Pharmaceutics 2020, 12, 16 12 of 16 Pharmaceutics 2020, 12, x 12 of 16 NCs can be clearly imaged with a fluorescence confocal microscope and they show a very good photostability while while good photostability using 10% usingof10% laserofpower, which is laser power, idealisforideal which the observation of in vitro for the observation of cellular in vitro uptake cellular(Figure 6C). The6C). uptake (Figure strategy used here The strategy usedconfers intrinsic here confers photoluminescence intrinsic to theto photoluminescence PLGA NCs the PLGA without introducing any cytotoxic quantum dots or photo-bleaching organic NCs without introducing any cytotoxic quantum dots or photo-bleaching organic dyes when dyes when compared to other more compared conventional to other approachesapproaches more conventional that physically thatblend imaging physically probes blend within imaging the carrier probes withinthat the can leadthat carrier to misinterpretations on the tracing can lead to misinterpretations on of thethe carrierof[12,13], tracing which the carrier may greatly [12,13], expand which may the greatly applications of this drug of expand the applications carrier. this drug carrier. Figure 6. Figure 6. (A) (A)Excitation Excitationandandemission emissionspectra spectraofofthe thenanocapsules nanocapsules (NCs) (NCs) water watersuspension; suspension; (B) fluorescence intensity of different concentrations of NCs measured by microplate (B) fluorescence intensity of different concentrations of NCs measured by microplate reader reader (n = 2, values (n = 2,represent mean ± sd and values represent mean subtract ± sdvalues of controlvalues and subtract non-fluorescence of controlNCs); and, (C) photostability non-fluorescence NCs); evaluation of NCs under confocal and, (C) photostability evaluationmicroscope of NCsatunder 10% laser power,microscope confocal fluorescence at intensity expressed 10% laser power,as the percentage vs. the value at the initial time, inset: NC1 water suspension observed fluorescence intensity expressed as the percentage vs. the value at the initial time, inset:at 60× lens. NC1 water suspension observed at 60× lens. Phantom studies were conducted to confirm the MRI performance of the capsules. Phantoms of NCs that were dispersed in agarose gel at various concentrations were prepared (Figure 7A). Phantom studies were conducted to confirm the MRI performance of the capsules. Phantoms of Spin-spin relaxation time (T2) maps clearly exhibit signal decay in a concentration dependent manner. NCs that were dispersed in agarose gel at various concentrations were prepared (Figure 7A). Spin- The calculated transverse relaxivity (r2 ) values at 7 Tesla of both non-PEGylated NC1 (263 mM−1 s−1 ) spin relaxation time (T2) maps clearly exhibit signal decay in a concentration dependent manner. The and PEGylated NC2 (237 mM−1 s−1 ) are similar as those seen in Figure 7B, as expected,−1 further calculated transverse relaxivity (r2) values at 7 Tesla of both non-PEGylated NC1 (263 mM s−1) and demonstrating the similar loading and distribution of SPIONs in the polymer shell matrix for both PEGylated NC2 (237 mM−1s−1) are similar as those seen in Figure 7B, as expected, further systems. When compared with other clinically used SPIONs systems, such as Feridex (98 mM−1 s−1 ) demonstrating the similar loading and distribution of SPIONs in the polymer shell matrix for both and Resovist (151 mM−1 s−1 ) [25], the much higher r2 value of our NCs formula is expected to be useful systems. When compared with other clinically used SPIONs systems, such as Feridex (98 mM−1s−1) for in vivo MRI tracking of the NCs. and Resovist (151 mM−1s−1) [25], the much higher r2 value of our NCs formula is expected to be useful for in vivo MRI tracking of the NCs. Pharmaceutics 2020, 12, 16 13 of 16 Pharmaceutics 2020, 12, x 13 of 16 Pharmaceutics 2020, 12, x 13 of 16 Figure 7. (A) T2 maps of a series of concentrations of NC1 in agarose phantoms; (B) r2 Figure Figure 7.7. (A) (A)T2T2maps maps of of a series a series of concentrations of concentrations ofinNC1 of NC1 in agarose agarose phantoms; phantoms; (B) r2 (B) r2 relaxivity relaxivity evaluation for the NCs. relaxivity evaluation evaluation for the NCs. for the NCs. Cell Viability 3.4. Cell Viability after after NCs NCs Uptake Uptake 3.4. Cell Viability after NCs Uptake Photoluminescent NCs Photoluminescent NCswere wereincorporated incorporated by by brain endothelial brain cells after endothelial cells several hours in after several culture, hours in Photoluminescent NCs were incorporated by brain endothelial cells after several hours in as seen in culture, asFigure seen in8A, with cytoplasmic Figure localization 8A, with cytoplasmic of the NCsofinthe localization perinuclear structures compatible NCs in perinuclear structures culture, as seen in Figure 8A, with cytoplasmic localization of the NCs in perinuclear structures with Golgi with compatible bodies andbodies Golgi endosomes. This subcellular and endosomes. localization This subcellular was confirmed localization by Z-stack was confirmed images by Z-stack compatible with Golgi bodies and endosomes. This subcellular localization was confirmed by Z-stack (Figure (Figure images 8B). Importantly, this cellular 8B). Importantly, uptakeuptake this cellular was biocompatible for endothelial was biocompatible cells, as for endothelial theas cells, main the images (Figure 8B). Importantly, this cellular uptake was biocompatible for endothelial cells, as the exposed cells during NCs circulation in blood vessels, since viability tests did not show main exposed cells during NCs circulation in blood vessels, since viability tests did not show signs signs of cell main exposed cells during NCs circulation in blood vessels, since viability tests did not show signs toxicity of at a wide cell toxicity at arange wideofrange NCs concentrations up to 500 of NCs concentrations to 500(Figure upµg/mL μg/mL8C,D) (Figureand8C,D) 48 h and exposure. 48 h of cell toxicity at a wide range of NCs concentrations up to 500 μg/mL (Figure 8C,D) and 48 h Only extremely exposure. Only high doses high extremely (1000doses µg/mL) withμg/mL) (1000 noticeable withoccupying noticeablespace difficulties occupying fordifficulties space cell culturing for exposure. Only extremely high doses (1000 μg/mL) with noticeable occupying space difficulties for showed a significant reduction in cell viability and number. cell culturing showed a significant reduction in cell viability and number. cell culturing showed a significant reduction in cell viability and number. Figure 8. NCsuptake uptake and cytotoxicity in human brain endothelial cells. (A) Representative Figure 8. Figure 8. NCs NCs uptakeand andcytotoxicity cytotoxicityin human in humanbrain endothelial brain cells. cells. endothelial (A) Representative images (A) Representative images of hCEMC/d3 cells stained with membrane dye PHK26 and exposed to 50 µg/mL of NC1 for 24of of hCEMC/d3 cells stained with membrane dye PHK26 and exposed to 50 μg/mL images of hCEMC/d3 cells stained with membrane dye PHK26 and exposed to 50 μg/mL ofh NC1 for 24 h (63× magnification); (63× magnification); (B) Orthogonal (B) Orthogonal view view of a Z-stack of of a Z-stack Propidium Iodideof(PI) Propidium Iodide stained cells (PI) (showing NC1 for 24 h (63× magnification); (B) Orthogonal view of a Z-stack of Propidium Iodide (PI) stained cells (showing the cell nuclei) the cell nuclei) and the fluorescent and the NCs; (C,D) fluorescent hCEMC/d3 cellsNCs; were(C,D) treatedhCEMC/d3 for 48 h withcells were different stained cells (showing the cell nuclei) and the fluorescent NCs; (C,D) hCEMC/d3 cells were treated for 48 hofwith concentrations NC1different concentrations and cell viability of NC1 with was determined and cell viability WST-8 wasassay reduction determined with or tripsinized treated for 48 h with different concentrations of NC1 and cell viability was determined with WST-8 reduction and counted assay orchamber, in a Neubauer (n =and tripsinized 3–4, counted in a Neubauer values represent ** p < (n chamber, mean ± SEM, = 3–4, values 0.01). WST-8 reduction assay or tripsinized and counted in a Neubauer chamber, (n = 3–4, values represent mean ± SEM, ** P < 0.01). 3.5. Protein Loading represent meanand In Vitro ± SEM, ** PRelease < 0.01). ProteinLoading 3.5. Protein loading and and In encapsulation Vitro Release efficiency were determined by lysing NCs with DMSO and 3.5. Protein Loading measuring and Incontent. total protein Vitro Release The BSA loading content was determined as ~10 µg BSA/mg NCs Protein (1 wt%) loading withloading and an EE%and encapsulation of around efficiency 40% forefficiency were determined both NC2.PEGylated by lysing NCs with DMSO and NC1.non-PEGylated and systems, as Protein encapsulation were determined by lysing NCs with DMSO and measuring total protein content. The BSA loading content was determined as ~10 μg BSA/mg NCs (1 measuring total protein content. The BSA loading content was determined as ~10 μg BSA/mg NCs (1 wt%) with an EE% of around 40% for both NC2.PEGylated and NC1.non-PEGylated systems, as wt%) with an EE% of around 40% for both NC2.PEGylated and NC1.non-PEGylated systems, as listed in Table 1, which indicated that the incorporation of PEG does not affect the protein encapsulation process. Note that a protein loading of 1 wt% is much higher than other reported values (0.03 wt% of vessel endothelial growth factor (VEGF) loaded PLGA NCs) [22] and the EE% of 40% is comparable to the PLGA NCs loaded with neurotrophin-3 or brain-derived neurotrophic factor (47%) Pharmaceutics [26]. 2020, 12, 16 14 of 16 BSA-loaded NCs were able to release protein cargo over time at physiological temperature in PBS media (32% protein release in one week). Figure 9A shows a fast BSA release within the first listed hours,inbut Table not1,after which indicated one thatcould day, which the incorporation be related toofthe PEG does not protein affect the protein degradation encapsulation in ex vivo conditions process. Note that a protein loading of 1 wt% is much higher than other of our assays. The amount of released protein in one week was similar when indirectly reported values (0.03 wt% of measured vessel from theendothelial growthprotein pellet retained factor (VEGF) (Figure loaded PLGA NCs) 9B,C), although the[22] and profile release the EE% of 40%aismore showed comparable sustained to the PLGA NCs loaded with neurotrophin-3 or brain-derived neurotrophic pattern over time, which could be associated to the protein that was trapped within the PLGAfactor (47%) [26]. BSA-loaded NCs were able to release protein cargo over time at physiological temperature in PBS polymer. mediaBy(32%usingprotein releaseprotein the model in one week). BSA, hereFigure we 9A shows were ableato fast BSA the prove release within loading the firstand capacity hours, but release not after one day, which could be related to the protein degradation in ex vivo conditions kinetics of the drug carrier, yet the preservation of protein functionality after the encapsulation of our assays. The amount process of released still needs to be protein in oneNevertheless, investigated. week was similar when indirectly in a previous work, wemeasured have shownfromthatthe VEGF pellet retained protein (Figure 9B,C), although the release profile showed a more could be encapsulated following a similar route and VEGF effect on cell proliferation couldsustained pattern over time, be which could be associated to the protein that was trapped determined [22], which implied the preserved protein functionality. within the PLGA polymer. Figure9.9.Protein Figure Protein release release temporal temporal profileprofile of the of the NCs. NCs. (A–C) (A)into NC1 (C) PBS (10NC1 in PBS mg/mL) were(10 mg/mL) incubated at 37 ◦ C in were incubated at 37 rotation and the°C in rotation released and the released BSA quantified as total BSA mass quantified as total(A) of protein released mass of protein together with released BSA (A)intogether content with NCs the remaining BSApellet content in the lysis by DMSO remaining (B) and NCs pellet by the released BSA DMSO lysisindirectly calculated (B) and the released from BSA calculated the pellet values indirectly (C). The percentage wasfrom the pellet calculated valuesunreleased versus intact (C). TheNCspercentage lysated alsowas in DMSO (n = 8, calculated valuesintact versus represent mean ± NCs unreleased SEM).lysated also in DMSO (n = 8, values represent mean ± SEM). By using the model protein BSA, here we were able to prove the loading capacity and release kinetics of the drug carrier, yet the preservation of protein functionality after the encapsulation process 4. Conclusions still needs to be investigated. Nevertheless, in a previous work, we have shown that VEGF could be We here following encapsulated transformaPLGA similarnanocapsules route and VEGF(NCs) into effect ona cell highly sensitive,could proliferation MRI/photoluminescence be determined [22], dual-modal imaging theranostic platform for which implied the preserved protein functionality. drug delivery by integrating the biocompatible and photoluminescent polyester BPLP into the PLGA molecular structure, as well as by incorporating 4. Conclusions superparamagnetic iron oxide nanoparticles (SPIONs). Furthermore, we have shown that PEGylation provides a hydrophilic surface to the NCs slowing down their flocculation rated and without We here transform PLGA nanocapsules (NCs) into a highly sensitive, MRI/photoluminescence modiying the size, SPIONs content or protein loading capacity of the NCs. In all cases, the functional dual-modal imaging theranostic platform for drug delivery by integrating the biocompatible and moieties are embedded in the PLGA shell with minimal interferences between them or with the photoluminescent polyester BPLP into the PLGA molecular structure, as well as by incorporating therapeutic protein. The developed magnetic PLGA-BPLP NCs show biocompatibility in vitro. In this superparamagnetic iron oxide nanoparticles (SPIONs). Furthermore, we have shown that PEGylation regard, the NCs did not affect the viability of endothelial cells in culture for concentration up to 500 provides a hydrophilic surface to the NCs slowing down their flocculation rated and without modiying μg/mL and 48 h incubation. Finally, we have shown that the NCs can contain 1 wt% of protein in the size, SPIONs content or protein loading capacity of the NCs. In all cases, the functional moieties their core achieved at fabrication level and that one third of the encapsulated protein is released in are embedded in the PLGA shell with minimal interferences between them or with the therapeutic the first week. Interestingly, the NCs decorated with SPIONs can be exploited for magnetic retention protein. The developed magnetic PLGA-BPLP NCs show biocompatibility in vitro. In this regard, and magnetic guiding. the NCs did not affect the viability of endothelial cells in culture for concentration up to 500 µg/mL and 48 h incubation. Finally, we have shown that the NCs can contain 1 wt% of protein in their core Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: achieved at fabrication level and that one third of the encapsulated protein is released in the first UV-Vis absorbance spectra and fluorescence spectra of the as-synthesized A/B) BPLP, C/D) PLGA- week. Interestingly, the NCs decorated with SPIONs can be exploited for magnetic retention and BPLP and E/F) reference anthracene at a series concentrations for fluorescence quantum yield magnetic guiding. Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/1/16/s1. Figure S1: UV-Vis absorbance spectra and fluorescence spectra of the as-synthesized A/B) BPLP, C/D) PLGA-BPLP and E/F) reference anthracene at a series concentrations for fluorescence quantum yield calculation, Figure S2: Matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF-MS) was used to determine the average molecular weight (Mw) of BPLP. Average Mw calculated from the mass spectrum of BPLP Pharmaceutics 2020, 12, 16 15 of 16 is 1044 g/mol. For the analysis, the sample was spotted in a MALDI plate with 1,8,9-Antracenotriol (Ditranol) and analyzed using a mass spectrometer (Bruker Daltonics Ultraflex TOF/TOF) in reflectron mode, Figure S3: SEM images and DLS size distributions of NCs fabricated by different types of PLGA-BPLP, Figure S4: Fluorescence intensity of different concentrations of NCs measured by microplate reader at the maximal emission wavelengths (values have subtracted values of control non-fluorescence NCs), Figure S5: Hysteresis loop (300 K) and ZFC-FC (inset) measurements of the lyophilized nanocapsule batch NC2. Author Contributions: Conceptualization, Y.Z. and A.R. (Anna Roig); Data curation, Y.Z. and M.G.-G.; Formal analysis, all authors; Investigation, Y.Z. and M.G.-G.; Methodology, Y.Z. and M.G.-G.; Resources, A.R. (Anna Rosell) and A.R. (Anna Roig); Supervision, A.R. (Anna Rosell) and A.R. (Anna Roig); Writing—original draft, Y.Z.; Writing—review & editing all authors. All authors have read and agreed to the published version of the manuscript. Funding: This work was partially supported by the Spanish Ministry of Science, Innovation and Universities through the grants PCIN-2017-090, RTI2018-096273-B-I00, CTQ2017-87637-R, SAF2017-87670-R, SEV-2015-0496 and MDM-2017-0720 and by the Generalitat de Catalunya grants (2017SGR765 & 2017SGR1427). Yajie Zhang was supported by the China Scholarship Council (CSC). The Acciones Complementarias program from the Instituto de Salud Carlos III, Spain, co-financed by the European Regional Development Fund (AC17/00004 grant) as part of the MAGBBRIS project (Euronanomed III 8th joint call), and the RETICS INVICTUS RD16/0019/0021 were acknowledged. Miguel Servet program (CPII15/00003) from Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III and ERDF, supported A. Roig research contract. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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