Nano-Assemblies from Amphiphilic PnBA-b-POEGA Copolymers as Drug Nanocarriers

The focus of this study is the development of highly stable losartan potassium (LSR) polymeric nanocarriers. Two novel amphiphilic poly(n-butyl acrylate)-block-poly(oligo(ethylene glycol) methyl ether acrylate) (PnBA-b-POEGA) copolymers with different molecular weight (Mw) of PnBA are synthesized via reversible addition fragmentation chain transfer (RAFT) polymerization, followed by the encapsulation of LSR into both PnBA-b-POEGA micelles. Based on dynamic light scattering (DLS), the PnBA30-b-POEGA70 and PnBA27-b-POEGA73 (where the subscripts denote wt.% composition of the components) copolymers formed micelles of 10 nm and 24 nm in water. The LSR-loaded PnBA-b-POEGA nanocarriers presented increased size and greater mass nanostructures compared to empty micelles, implying the successful loading of LSR into the inner hydrophobic domains. A thorough NMR (nuclear magnetic resonance) characterization of the LSR-loaded PnBA-b-POEGA nanocarriers was conducted. Strong intermolecular interactions between the biphenyl ring and the butyl chain of LSR with the methylene signals of PnBA were evidenced by 2D-NOESY experiments. The highest hydrophobicity of the PnBA27-b-POEGA73 micelles contributed to an efficient encapsulation of LSR into the micelles exhibiting a greater value of %EE compared to PnBA30-b-POEGA70 + 50% LSR nanocarriers. Ultrasound release profiles of LSR signified that a great amount of the encapsulated LSR is strongly attached to both PnBA30-b-POEGA70 and PnBA27-b-POEGA73 micelles.


Introduction
Nano-drug delivery systems (NDDSs) have revolutionized drug delivery in the last few decades, endowing drugs with increased stability and water solubility, prolonging the cycle time, enhancing the uptake rate of target cells or tissues, reducing enzyme degradation, and controlling the release of the drug [1][2][3][4].
The NDDSs used in modern biomedicine concern supramolecular self-assembled structures divided into organic, inorganic, and composite materials. Amphiphilic block copolymers (AmBCs) have been the workhorse of pharmaceutical nanotechnology in recent years towards the design of drug-loaded block copolymer nanocarriers for the demanding treatment of crucial diseases [5][6][7][8]. The delivery of drugs, proteins, and nucleotides is possible via AmBCs self-assembled into biocompatible nanostructured multifunctional structures such as micelles, polymer containing liposomes, or polymeric nanoparticles [9].
A multitude of applications in nanomedicine and diagnostics are powered by Am-BCs [10][11][12]. The unique core-shell architecture of AmBC nanoassemblies enables a physical encapsulation of drug molecules into their hydrophobic micellar core, conferring an enhanced drug solubility and reduced toxicity. The hydrophilic shell stabilizes the core, The stability in fetal bovine serum (FBS) of PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles was examined by dynamic light scattering (DLS) to gain further insight into the protein-polymer interactions. A plethora of NMR experiments were performed to explore the intramolecular interactions between the PnBA 30 -b-POEGA 70 micelles and LSR, and to discover the self-diffusion coefficients D of the PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers. Moreover, temperature studies using 1 H-NMR spectroscopy for the POEGA hydrophilic homopolymer and the PnBA-b-POEGA micelles over the temperature range 25-80 • C were further conducted to trace the mobility of protons located in the hydrophobic PnBA micellar core. At last, drug loading (DL%) and encapsulation efficiency (%EE) were calculated by means of ultraviolet visible (UV-Vis) spectroscopy, followed by the ultrasound-triggered release of LSR from PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles.

Synthesis of PnBA-b-POEGA Diblock Copolymers
The hydrophobic PnBA homopolymers were synthesized in 4000 and 7800 g/mol M w variations in a previous work, and used as macro-CTAs for commencing the subsequent polymerization of the OEGA monomer [38].
The synthesis of PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 block copolymers involved AIBN as the initiator, PnBA (4000 and 7800 g/mol) homopolymers as the macro-CTAs, and 1,4-dioxane as the solvent. A typical RAFT polymerization procedure for the synthesis of the PnBA 30 -b-POEGA 70 diblock in 4000 molar mass of PnBA is described as follows: to a round-bottom flask, 25 mL, PnBA (0.38 g), AIBN (0.00314 g, 0.019 mmol), OEGA (480 g/mol, 0.89 g), and 6.4 mL of 1,4-dioxane were added with a magnetic stirrer and fitted with a septum. The final solution was degassed by nitrogen gas bubbling for 15 min and placed in a thermostatted oil bath at 70 • C for 24 h. After the polymerization reaction, the product was precipitated in 10-fold excess of n-hexane and dried in a vacuum oven for 48 h (yield: 65%). The dry PnBA 30 -b-POEGA 70 diblock was collected and stored.

Preparation of Self-Assembled PnBA-b-POEGA Diblock Copolymers
Despite the fact that PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 block copolymers are dissolved directly in aqueous media, their micelles were prepared using the thin film hydration method (TFHM). The contradiction is explained as LSR-loaded nanocarriers were also prepared with TFHM (more details are given in Section 2.5). Specifically, 10 mg of each diblock was dissolved in ACTN (stock solution) and allowed to stand for polymer dissolution. Afterwards, each polymer stock solution was transferred into a flask and placed in a rotary evaporator for the efficient evaporation of ACTN until a thin film of each polymer was formed around the inner part of the flask. When the thin film was formed, 10 mL filtered water for injection (WFI) was added to the flask and stirred until the entire thin film was dissolved. The concentration of the aqueous stock solutions was 10 −3 g/mol.

FBS Interactions with PnBA-b-POEGA Diblock Copolymers
The mixtures of PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 copolymers with clarified FBS were prepared in filtered PBS using two protocols based on different polymer dilution to FBS and different FBS:PBS ratios. The copolymer stock solutions were prepared with direct dissolution in filtered WFI at a concentration of 3 × 10 −3 g/mL for both protocols. According to the first protocol, 50 µL of each diblock was mixed with 3 mL FBS:PBS (1/9 v/v) ratio. The second protocol included the mixing of 100 µL sample with (a) 3 mL FBS:PBS (1/9 v/v) ratio and (b) 3 mL FBS:PBS (1/1 v/v). All FBS-copolymer mixtures were filtered through 0.45 µm pore size filters and allowed to stand 1 h for equilibration before DLS measurements.

Preparation of LSR-Loaded PnBA-b-POEGA Nanocarriers
The LSR-loaded PnBA 30 -b-POEGA 70 + 20% LSR, PnBA 30 -b-POEGA 70 + 50% LSR and PnBA 27 -b-POEGA 73 + 20% LSR, PnBA 27 -b-POEGA 73 + 50% LSR nanocarriers were prepared using the TFHM. Specifically, an appropriate amount of LSR was dissolved in ACTN to prepare 20 wt.% and 50 wt.% concentrations of LSR in each copolymer-drug mixture. Afterwards, each copolymer and LSR stock solution was mixed in the appropriate amounts. Each copolymer-drug mixture was transferred into a flask and placed in a rotary evaporator for the efficient evaporation of ACTN until a thin film of each copolymer and LSR was formed around the inner walls of the flask. Then, 5 mL filtered WFI was added to the flask and stirred until the entire thin film was dissolved. The concentrations of the aqueous stock solutions were 10 −3 g/mol. All copolymer and drug-loaded solutions were filtered through 0.45 µm pore size filters and allowed to stand overnight for equilibration before measurements. A schematic illustration for the preparation of LSR-loaded PnBA-b-POEGA nanocarriers using the TFHM is proposed in Scheme 1. Scheme 1. Schematic illustration for the preparation of LSR-loaded PnBA-b-POEGA nanocarriers using the TFHM in aqueous solutions.

Drug Loading and Encapsulation Efficiency Calculations of LSR
The percentage of LSR incorporated into PnBA 30 -b-POEGA 70 + 50% LSR and PnBA 27b-POEGA 73 + 50% LSR nanocarriers was estimated by UV-Vis (Perkin-Elmer, Lambda 19 spectrophotometer, Waltham, MA, USA) spectroscopy. LSR concentration was calculated with the aid of the following LSR calibration curve in ACTN, which was on the general form y = ax + b (where a is the slope and b is the intercept): LSR concentration (g/mL) = (y − 0.22504)/98,462.16599 (1) The absorbance (y) of the drug-polymer solutions was measured at 205 nm. %DL is the amount of drug loaded per unit weight of the micelle/nanoparticle, and is calculated by the amount of total entrapped drug divided by the total micelle/nanoparticle weight. %EE is the percentage of drug that is successfully entrapped into the micelle/nanoparticle, and is calculated by the total encapsulated drug divided by the total drug added. %DL %EE were calculated using the following Equations:

Ultrasound Release Studies
The release of LSR from PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles was studied in WFI in an ultrasonic bath sonicator for approximately five hours. Specifically, 5 mL of PnBA 30 -b-POEGA 70 + 50% LSR and PnBA 27 -b-POEGA 73 + 50% LSR nanocarriers were added in dialysis bags of 3.5 kDa MWDO. Afterwards, the dialysis bags were inserted into 100 mL filtered WFI and placed in a SOLTEC, SONICA 3300ETH-S3 ultrasonic bath. Aliquots of samples were taken from the external solution at specific time intervals and each time the aqueous solution was restored to its initial volume, so that the reservoir conditions remained constant. On the basis of the calibration curve of the Equation (1), the amount of LSR released at different time intervals, up to 5 h, was determined using UV-Vis (Perkin-Elmer, Lambda 19 spectrophotometer, Waltham, MA, USA) spectroscopy at λ max = 205 nm.

Evaluation
M w and M w /M n of the synthesized PnBA-b-POEGA block copolymers were determined by a size exclusion chromatography (SEC) instrument from Waters Technologies Corporation, Caguas, Puerto Rico. It was equipped with a Waters 1515 isocratic pump, a set of three µ-Styragel mixed bed columns (porosity range between 10 2 to 10 6 Å), and a Waters 2414 refractive index detector (equilibrated at 40 • C). Breeze software was utilized for data acquisition and analysis. THF containing 5% v/v triethylamine was the mobile phase at a flow rate of 1.0 mL/min at 30 • C. The setup was calibrated with linear polystyrene standards, having narrow M w /M n and weight average M w in the range of 1200 to 929,000 g/mol. Concentrations in the range 2-4 mg/mL were used for analysis.
Fluorescence spectroscopy (FS) experiments were pursued to determine the CMC of the PnBA-b-POEGA block copolymers using a Fluorolog-3 Jobin Yvon-Spex spectrofluorometer, model GL3-21 (HORIBA Scientific, Piscataway, NJ, USA) with pyrene as the fluorescent probe. A stock solution of 1 mM Py in ACTN was prepared and added to the solutions in a ratio of 1 µL per 1 mL polymer solution. The excitation wavelength used for the measurements was 335 nm and emission spectra were recorded in the region of 355-630 nm. Measurements were conducted after the evaporation of ACTN overnight at room temperature.
DLS measurements were performed using an ALV/CGS-3 Compact Goniometer System (ALV GmbH, Siemensstraße 4, 63225 Langen, Hessen, Germany) with an ALV-5000/EPP multi-τ digital correlator with 288 channels and an ALV/LSE-5003 light scattering electronic unit for stepper motor drive and limit switch control. A JDS Uniphase 22-mW He-Ne laser (632.8 nm) was used as the light source. The size data and figures provided in the manuscript are from averaged measurements at 90 degrees, (five measurements per concentration/angle). The obtained correlation functions were analyzed by the cumulants method and CONTIN software (ALVGmbH, Hessen, Germany). All solutions were filtered through 0.45 µm hydrophilic PTFE filters (Millex-LCR from Millipore, Billerica, MA, USA) before measurements. Static light scattering (SLS) measurements were carried out on the same instrument at the same temperature and angular range as in DLS measurements, using toluene as the calibration standard. All samples were prepared at a concentration of 1 × 10 −3 g/mL. SLS data were determined using Zimm plots.
A ZetaSizer Nanoseries Nano-ZS (Malvern Instruments Ltd., Malvern, UK) was used for the electrophoretic light scattering (ELS) measurements, equipped with a 4 mW solid-state laser at a wavelength of 633 nm and a fixed backscattering angle of 173 • . The

Synthesis and Molecular Characterization of PnBA-b-POEGA Diblock Copolymers
Two amphiphilic PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 diblock copolymers were synthesized with different M w of a PnBA block. The PnBA segment enables the formation of equilibrated micellar structures in aqueous media in most cases [39][40][41]. In the current study, the previously synthesized PnBA homopolymers (in 4000 and 7800 g/mol M w variations) [38] were used as macro-CTAs to commence the polymerization of the OEGA. The employment of DDMAT as the CTA leads to polymers with well-controlled M w and M w /M n [25,42,43]. The synthetic route of PnBA-b-POEGA copolymers using RAFT polymerization and the chemical structures of the diblocks and LSR are presented in Schemes 2 and 3. Polymerization of OEGA proceeded in 65% yield. The molecular characteristics of PnBA and PnBA-b-POEGA copolymers determined by SEC and 1 H-NMR spectroscopy are provided in Table 1. The obtained M w were in agreement with the calculated stoichiometric ones. The M w /M n values, reported in Table 1, fall within the range usually reported for RAFT synthesized homopolymers and block copolymers. A representative SEC chromatogram of 4000 g/mol PnBA and PnBA 30 -b-POEGA 70 diblock is depicted in Figure 1, highlighting the increase in M w after the addition of POEGA. The M w /M n are narrow, monomodal, and almost symmetrical, with a minimal number of tails appearing at high elution volumes (in the case of PnBA 30 -b-POEGA 70 diblock), indicating an almost integrated reinitiation of RAFT polymerization and a nearly clean extension of the block sequence.
The compositions of PnBA-b-POEGA diblock copolymers were verified by 1 H-NMR spectroscopy. A representative 1 H-NMR spectrum of the PnBA 30 -b-POEGA 70 diblock is presented in Figure 2. The calculated composition values are also summarized in Table 1. All data manifest a successful and controlled polymerization process.

Physicochemical Characterization of the PnBA-b-POEGA Micelles
Detailed physicochemical characterization is provided for the assessment of CMC, size, shape, surface charge, and chemical structure of PnBA-b-POEGA block copolymers by means of FS, DLS, SLS, ELS, and ATR-FTIR techniques. CMC was determined using the FS technique by entrapping Py (fluorescence probe) into the micellar core of PnBA-b-POEGA copolymers [see Section S1.1, Figure S1, Supplementary Materials]. Both CMC values are listed in Table 2. Based on the chain architecture and DLS/SLS data, the novel diblocks self-assembled into nanosized core-shell micelles in aqueous media with PnBA hydrophobic cores and a hydrophilic POEGA corona. A comparison of intensity weighted and monomodal size distribution plots of PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 diblocks is presented in Figure 3a. The two symmetrical peaks indicate the participation of all chains in the formation of block copolymer micelles. Higher M w of PnBA elicited an important growth in hydrodynamic radius (R h ), an almost sixfold higher scattered light intensity value (mass) and a rather increased aggregation number (N agg, obtained by SLS) of PnBA 27 -b-POEGA 73 diblock (red line) compared to PnBA 30 -b-POEGA 70 (black line), introducing an increase in the total M w of the system. The values of R h , scattered light intensity, polydispersity index (PDI), and N agg of the PnBA-b-POEGA diblock copolymers formed in aqueous media at C = 10 −3 g/mL, pH = 7, and 25 • C are summarized in Table 2. ELS measurements of the PnBA-b-POEGA copolymers in aqueous media revealed slightly negative ζ pot , as seen in Table 2, maybe due to the presence of carboxylic acid chain end groups from the CTA.

FBS Interactions with PnBA-b-POEGA Block Copolymers
The lack of stability of polymeric biomaterials in a biological environment could induce dramatic changes in their synthetic identity as a result of recurring chemical/physical interactions between the polymer and the medium components. Thus, DLS stability studies of amphiphilic PnBA-b-POEGA micelles in FBS solutions were performed to detect possible alterations of their physicochemical characteristics in terms of size, PDI, and scattered light intensity. FBS simulates the physicochemical conditions of blood, and contains a variety of proteins, including a substantial amount (approximately 2.5 mg/mL) of bovine serum albumin (BSA). It is considered to differentiate/elucidate the properties of the micelles when injected into the human plasma [21].  Table 3.
In terms of scattered light intensity (Table 3)     ELS measurements displayed strongly negative ζ pot values (Table 4) as LSR concentration increased from 20% to 50% for both PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles due to the negative charge of the encapsulated LSR. The values of R h , scattered light intensity, PDI, and ζ pot of the LSR-loaded PnBA-b-POEGA nanocarriers formed by TFHM in aqueous media are summarized in Table 4.
ATR-FTIR and UV-Vis measurements were pursued to examine LSR encapsulation into the polymeric core of the PnBA-b-POEGA micelles. A comparison of ATR-FTIR of neat PnBA 30 -b-POEGA 70 /PnBA 27 -b-POEGA 73 micelles and LSR-loaded PnBA-b-POEGA nanocarriers prepared by TFHM is provided in Figure 6a,b. A critical decline in ATR-FTIR intensity peaks is noticed after 20% and 50% LSR encapsulation, denoting significant structural changes in both copolymer-drug systems. Likewise, new characteristic peaks of LSR (green annotations) are clearly observed in the ATR-FTIR spectra of the nanocarriers. In both Figure 6a Figure 6b, which is attributed to the primary OH stretching vibrations of LSR [44,45]. All data confirm the existence of LSR in the mixed copolymer-drug aqueous solutions.
UV-Vis measurements further verified the successful encapsulation of LSR into the PnBA-b-POEGA polymeric micelles. Figure 7 presents the UV-Vis spectra of neat PnBA 30 -b-POEGA 70 /PnBA 27 -b-POEGA 73 micelles and LSR-loaded PnBA-b-POEGA nanocarriers prepared by TFHM in aqueous solutions along with the corresponding spectra of LSR in ACTN. In line with the literature data, the UV spectrum of LSR exhibits maximum absorbance at 205 nm, 225 nm, and 254 nm wavelengths [44,[46][47][48][49]. However, the UV-Vis spectra of LSR, depicted in Figure 7a (Figure 7b) nanocarriers is attributed to the LSR UV absorption peaks. As LSR concentration increases to 50% (blue lines), an increase in the UV absorption of LSR at both wavelengths is clearly evident, highlighting a greater/stronger LSR encapsulation into the micelles. Notably, the detected shifting in the peak maxima may be related to hydrophobic and hydrogen bonding copolymers/drug interactions.

Stability Studies of LSR-Loaded PnBA-b-POEGA Nanocarriers
Stability studies were performed using DLS to estimate the temporal stability of the resulting LSR-loaded PnBA-b-POEGA solutions over a period of 23 days. The R h and scattered intensity measurements versus time (average of three measurements) of LSR-loaded PnBA-b-POEGA nanocarriers are depicted in Figure 8. DLS studies on the mixed-drug solutions verified their stability in aqueous solutions in terms of size and mass. Specifically, the PnBA 30 -b-POEGA 70 + 20% LSR/PnBA 27 -b-POEGA 73 + 50% LSR nanocarriers (Figure 8a,b), as well as the PnBA 27 -b-POEGA 73 + 20% LSR/PnBA 27 -b-POEGA 73 + 50% LSR (Figure 8c,d), do not exhibit significant R h and intensity fluctuations, indicating stable nanostructures. Slight variations in scattered light intensity are present in the case of PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers (Figure 8b), suggesting an appreciably stable drug-polymer system within 23 days. Remarkably, the mixed copolymer-drug solutions exhibited great long-term stability to the naked eye (approximately 2 years), as no visually detectable drug precipitation was observed.    Figure 10 shows the expected proton signals of LSR, verifying the successful loading of LSR into the polymeric nanocarriers. The chemical shifts of the PnBA 30 -b-POEGA 70 proton signals in the absence (Figure 9) and in the presence of LSR ( Figure 10) are summarized in Table 5. Additionally, the chemical shifts of LSR proton signals in the presence of PnBA 30 -b-POEGA 70 copolymer and in sodium dodecyl sulfate (SDS) micelles are exhibited in Table 6, reflecting its great flexibility [32].      A shielding of the copolymer resonances is shown in the presence of LSR, evidencing the successful loading of LSR in the micellar environment. LSR chemical shifts are also driven to upfield values, reaching ∆δ differences of up to 0.19 ppm, indicating its strong interactions with the copolymer structure.

2D-COSY Studies on LSR-Loaded PnBA-b-POEGA Nanocarriers
The intramolecular interactions between the PnBA 30 -b-POEGA 70 copolymer and LSR moieties and their internal structure were evaluated by 2D-COSY measurements. The 2D-COSY spectrum of the PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers is presented in Figure 11, where the black arrows denote the protons of the copolymer structure while the red ones point to the LSR peaks. The cross peaks, noticed between the protons of the copolymer and the protons of LSR in the 2D-COSY spectrum in Figure 11, signify the intramolecular interactions and the structural elucidation of both copolymer and LSR.

2D-NOESY Studies on LSR-Loaded PnBA-b-POEGA Nanocarriers
The intramolecular/intermolecular interactions between the copolymer and LSR were further investigated via 2D-NOESY experiments for the verification of the successful loading of LSR into the polymeric PnBA 30 -b-POEGA 70 micelles, and to study the spatial vicinities between the copolymer and LSR. The eminent correlations between PnBA 30 -b-POEGA 70 and LSR moieties in D 2 O are juxtaposed in Figure 12. The cross peaks between the phenyl rings (8-9, 10-11, 12-13, 14-15) and butyl chain of LSR (1,2,3,4) with the methylene signals of PnBA (c, d, e, f) are clearly evident (Figure 12), highlighting the strong binding of LSR with the PnBA hydrophobic core. Figure 12b illustrates the approach of LSR in PnBA 30 -b-POEGA 70 micelles. Particularly, the correlations between the butyl chain (1,2,3,4) of LSR and the butyl chain (c, d, e, f) of PnBA seem to be more eminent than those between the biphenyl ring (8-9, 10-11, 14-15, 12-13) of LSR and the butyl chain (c, d, e, f) of PnBA, evidencing that the hydrophobic interactions are mainly exerted by the butyl chain of LSR and the butyl chain of PnBA.  Figure 13, and may be related to the diffusions of block copolymer micelles and unimolecular block copolymers, respectively. The trace with the highest constant (D = 4.24 × 10 −10 m 2 s −1 ) depicted in Figure 13, may refer to the diffusion of LSR, suggesting a rather high exchange between the free and the micelle-bound state of LSR.  Figure 14) with increasing temperature, which is an indication of enhanced proton mobility. The half-width of POEGA is decreased, while the integrated intensity of the proton signals fluctuates from 25 • C to 80 • C. Specifically, the integrated intensity of the proton signals steeply raises as temperature increases from 25 • C to 55 • C. The subsequent decrease in the integrated intensity of the proton signals for temperatures from 55 • C to 80 • C signifies a small decrease in its solubility, which may be exerted by the rupture of hydrogen bonds upon heating. The 1 H-NMR peaks of PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 diblock copolymers presented in Figures 15 and 16 shifted with increasing temperature. At high temperatures (80 • C), the proton signals appear narrower and more intense, implying the enhanced mobility of the protons. The chemical shifting as a function of temperature of PnBA 30 -b-POEGA 70 block copolymers is presented in Figure S4 in the Supplementary Materials to provide a clearer view of the shifting of the 1 H-NMR spectra ( Figure 15) to the lower field regions.
Apparently, the novel PnBA-b-POEGA copolymers are considered temperature-indepe ndent AmBCs, containing a biocompatible and rather hydrophobic easily deformable PnBA segment [50]. Based on the low T g value (−54 • C) of PnBA [46,51], heating may trigger partial disintegration or increased fluidity of the polymer core matrix by inducing water penetration in the PnBA core, resulting in increased proton mobility. The effect of temperature on PnBA 30 -b-POEGA 70 block copolymers was also investigated by DLS measurements to provide useful information of the overall hydrodynamic behavior of the globular structures. DLS data analysis details are presented in Section S1.3, Figure S3, Supplementary Materials.

Encapsulation and Ultrasound Release Studies
DL% and %EE were calculated by means of UV-Vis spectroscopy. The ultrasoundtriggered release behavior of LSR from PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles was also considered. The highly stable PnBA 30 -b-POEGA 70 + 50% LSR and PnBA 27 -b-POEGA 73 + 50% LSR nanocarriers were hand-picked for the investigation of release kinetics, mainly due to the increased concentration of hydrophobic LSR (50%). The %DL and %EE values were calculated using Equations (2) and (3), and are summarized in Table 7. The highest hydrophobicity of the PnBA 27 -b-POEGA 73 micelles, emanating from the increased M w of the PnBA block (7800), seems to favor the encapsulation of hydrophobic LSR into the micelles, resulting to a higher value of %EE compared to PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers ( Table 7). Given that PnBA is not considered a highly hydrophobic polymer (taking also into account the low T g value) [46] in combination with the low M w of the PnBA block (4000), the PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers exhibited low %EE, implying an inefficient loading of LSR into PnBA 30 -b-POEGA 70 micelles. The cumulative drug release percentage vs. time is provided in Figure 17 for the PnBA 30 -b-POEGA 70 + 50% LSR (black line) and PnBA 27 -b-POEGA 73 + 50% LSR (red line) nanocarriers. The release profile of LSR started simultaneously with the application of ultrasound, exhibiting a gradual increase in released LSR within five hours during the release experiments. Based on the UV-Vis results in Figure 17 for both nanocarriers, an initial burst release of LSR is observed in the first ten minutes, followed by a relatively gradual increase of released LSR until reaching a plateau. The relatively low LSR release (Table 7) implies that a large portion of the encapsulated LSR is tightly bound to the cores of polymeric PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles, and it is quite difficult to release.

Conclusions
Novel biocompatible PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 AmBCs were successfully synthesized in different M w of the PnBA block using RAFT polymerization, and highly stable drug-loaded nanocarriers were developed through TFHM. The PnBA-b-POEGA diblocks and biocompatible drug-loaded nanocarriers (formed by the encapsulation of LSR into amphiphilic PnBA-b-POEGA copolymers) were studied using a wide range of physicochemical methods.
The PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 diblock copolymers self-assembled into nano-sized core-shell micelles in aqueous milieu. The engulfing of LSR into the inner hydrophobic segments of copolymers contributed to the formation of greater size drug-polymer nanostructures.
Likewise, FBS interactions with PnBA-b-POEGA copolymers indicated a good stability of polymeric micelles in a biological environment, endorsing the idea that POEGA hydrophilic chains shield the copolymer micelles against protein adsorption and agglomeration. DLS stability studies for the LSR-loaded PnBA-b-POEGA nanocarriers suggested highly stable drug-polymer systems for a period of 23 days.
ATR-FTIR measurements verified the existence of LSR in the mixed copolymer-drug aqueous solutions by detecting new characteristic peaks of LSR in the ATR-FTIR spectra of the LSR-loaded PnBA-b-POEGA nanocarriers. Moreover, UV-Vis experiments also confirmed the successful encapsulation of LSR into the PnBA-b-POEGA micelles. 1 H-NMR measurements further corroborated the presence of LSR into PnBA 30 -b-POEGA 70 micelles in aqueous media. 2D-COSY, 2D-NOESY, and 2D-DOSY experiments confirmed the internal structure of the PnBA 30 -b-POEGA 70 copolymer and LSR moieties and documented the successful encapsulation of LSR into the micelles. Specifically, 2D-NOESY experiments evidenced the strong intermolecular association between the biphenyl ring and butyl chain of LSR structure with the methylene signals of PnBA. Interestingly, the PnBA 30b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles exhibited enhanced proton mobility, probably caused by partial loosening of the PnBA hydrophobic core upon temperature increase.
The highest hydrophobicity of the PnBA 27 -b-POEGA 73 micelles contributed to an efficient encapsulation of LSR into the micelles exhibiting a greater value of %EE compared to PnBA 30 -b-POEGA 70 + 50% LSR nanocarriers. Release rates of LSR implied that a significant amount of the encapsulated LSR is tightly bound to both PnBA 30 -b-POEGA 70 and PnBA 27 -b-POEGA 73 micelles, leading to relatively low release.
The current study presents an overall physicochemical characterization of novel block copolymer micellar carriers, including NMR measurements, which can accurately detect drug-copolymer intermolecular interactions in the system.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10 .3390/polym13071164/s1, Figure S1: comparative diagram of the calculated relative intensity ratio I1/I3 of pyrene peaks versus the copolymer concentration in water for the PnBA30-b-POEGA70 and PnBA27-b-POEGA73 copolymers; Figure

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.