Hybrid nanocomposites, obtaining process, pharmaceutical compositions and uses

Abstract

The present technology relates to hybrid nanocomposites based on pldla-co-tmc@peo-ppo-peo and ferrofluid, which can be used for magnetic hyperthermia and drug delivery. The technology also relates to the process of obtaining the hybrid nanocomposites, to pharmaceutical compositions containing the hybrid nanocomposites, and to the use of the hybrid nanocomposites or compositions as drug delivery systems, especially for antitumor drugs.

Description

[01] The present technology relates to hybrid nanocomposites based on PLDLA-co-TMC@PEO-PPO-PEO and ferrofluid, which can be used for magnetic hyperthermia and drug delivery. The technology also relates to the process of obtaining the hybrid nanocomposites, to the pharmaceutical compositions containing the hybrid nanocomposites and to the use of the hybrid nanocomposites or compositions as drug delivery systems, especially antitumor drugs.

[02] The development of new advanced nanomaterials as a basis for drug delivery and use in hyperthermia creates new perspectives for therapy and other medical practices in oncology. The presence of magnetic nanoparticles allows their use in magnetic hyperthermia. This type of medical-therapeutic treatment is possible due to the behavior of magnetic nanoparticles when placed in an alternating current magnetic field, with the resulting heating, between 40°C and 45°C, being sufficient to destroy tumor cells.

[03] Magnetic iron oxide nanoparticles (magnetite, Fe3O4, or maghemite, γFe2O3, for example) have potential applications in biology and medicine due to their superparamagnetic behavior, high saturation magnetization, high magnetic susceptibility, and low biotoxicity. However, magnetic iron oxide nanoparticles exhibit chemical instability in aqueous media, can be oxidized or agglomerated, have a short circulation time in biological environments, and potentially have low affinity for target sites in the body, limiting their viability in diagnostic and therapeutic procedures. To solve these problems, coating magnetite or maghemite nanoparticles with biocompatible polymers has become a viable option. Petition 870190047245, dated 05 / 20 / 2019, page 15 / 44 2 / 20 a promising alternative for obtaining stable hybrid nanocomposites for biological or medical applications.

[04] The formulation and application of hybrid nanoparticles composed of polymeric and magnetic materials are of great interest for biological applications and in medical practices, due to the biocompatibility of the material, the minimal or virtual absence of biotoxicity, and the adequate biodegradability. Polymeric nanoparticles, based on polyester, have been used as nano-biomaterials for the encapsulation of magnetite, allowing the achievement of innovative physicochemical properties.

[05] The terpolymer poly(L-co-D,L lactic acid-co-trimethylene carbonate) (PLDLA-co-TMC) is a biodegradable polyester with a high molar mass, which is biocompatible and bioresorbable. On the other hand, poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide), or PEO-PPO-PEO, is an amphiphilic copolymer indicated for controlled-release nanosystems of hydrophobic drugs and promising in the coating and stabilization of nanosystems.

[06] In the state of the art, polymer-coated magnetic nanocomposites are described, as exemplified below.

[07] Wang, N and colleagues (2013) obtained, using the crosslinking method, nanoparticles with a magnetite core covalently coated with the PEO-PPO-PEO triblock copolymer. According to the authors, the covalent bonds between amino groups on the particle surface and the carboxylic groups of the polymer ensure the stability of the formulation. The obtained particles could be used as a sustained-release system for hydrophobic drugs. Maeng JH and colleagues (2010) synthesized Fe3O4 theranostic nanoparticles with a PEO-TMA-FA polymeric coating for doxorubicin delivery (Wang, N., Guan, Y., Yang, L., Jia, L., Wei, X., Liu, H., & Guo, C. (2013). Journal of Colloid and Interface Science Magnetic Petition 870190047245, dated 05 / 20 / 2019, page 16 / 44 3 / 20 nanoparticles ( MNPs ) covalently coated by PEO - PPO - PEO block copolymer for drug delivery. Journal of Colloid And Interface Science, 395, 50-57 / Hee, Jin., Lee, Don-haeng., Hee, Kyung., Bae, You-han., Park, Insuh., Jeong, Seok., Jeon, Yong-sun., Shim, Chang-koo., Kim, Wooyoung., Kim, Jungahn., Lee, Jeongmi., Lee, Yoon-mi., Kim, Ji-hee., Kim, Wonhong., Hong, Soon-sun (2010). Biomaterials Multifunctional doxorubicin loaded superparamagnetic iron oxide nanoparticles for chemotherapy and magnetic resonance imaging in liver cancer. Biomaterials, 31(18), 49955006).

[08] Patent documents BR102017002165-3, entitled “Nano spray drying for the production of theranostic platforms of iron oxide nanoparticles with polyoxyethylene-polyoxypropylene block copolymer (PEO-PPO-PEO)”, filed on 01 / 02 / 2017 and WO2019006440, entitled “Magnetic nanoparticles for targeted delivery”, priority date 30 / 06 / 2017, describe iron nanoparticles coated with PEO-PPO-PEO.

[09] The hybrid nanocomposite of the present invention is based on the terpolymer PLDLA-co-TMC, the block copolymer PEO-PPO-PEO and the magnetic ferrofluid FeXOY; FeXOY being coated by the polymeric matrix PLDLA-co-TMC@PEO-PPO-PEO.

[010] The process for obtaining hybrid nanocomposites using this technology allows the production of polymeric nanoparticles with an average hydrodynamic diameter between 50 nm and 300 nm. This diameter range enables the internalization of the nanoparticles into neovascularized tissues, which is the case for tumors such as benign prostatic hyperplasia. Petition 870190047245, dated 05 / 20 / 2019, page 17 / 44 4 / 20

[011] The combination of polymers, resulting in poly(L-co-D,L lactic acid-co-trimethylene carbonate)@poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) [(PLDLA-co-TMC)@PEO-PPO-PEO], for the encapsulation of iron oxide nanoparticles, allows the development of better physicochemical properties of hybrid nanocomposites (FeXOY@PLDLA-CO-TMC@PEO-PPO-PEO) for biomedical applications, compared to other similar nanosystems.

[012] The solvent displacement method used in the process described in this technology allows for the production of low-cost polymeric nanoparticles (PLDLA-co-TMC@PEO-PPO-PEO), as well as ease of processing and encapsulation efficiency. Due to the miscibility of the solvents in the organic (internal) and aqueous (external) phases, which determines the nucleation, growth, and aggregation conditions of the nanoparticles, effective encapsulation of hydrophobic and hydrophilic drugs, as well as magnetic nanoparticles such as magnetite or maghemite, is possible.

[013] The proposed process, involving solvent displacement and joining of PLDLA-co-TMC to PEO-PPO-PEO for FeXOY encapsulation, demonstrated effectiveness in preserving the chemical-structural characteristics of the FeXOY magnetic ferrofluid, preventing agglomeration and oxidation, in addition to allowing the obtaining of a hybrid nanocomposite with the ability for controlled drug release.

[014] The hybrid nanocomposite of the present technology is biocompatible, stable, has a high molar mass polyester coating that protects the magnetic nanoparticles, such as magnetite (Fe3O4) or maghemite (Fe2O3), from oxidation, has potential for medical therapy in oncology based on magnetic hyperthermia, and exhibits susceptibility to the release of drugs (antineoplastics, for example), usually with varying levels of toxicity to normal human body tissue. The nanocomposite of the present technology can Petition 870190047245, dated 05 / 20 / 2019, page 18 / 44 5 / 20 serve as a nanocarrier for lipo- or hydrosoluble drugs with characteristics suitable for intracellular internalization in diseased tissues and / or organs, such as cancer. The hybrid nanocomposite containing a drug, if administered orally or intravenously, can be locally targeted to the tumor by magnetic hyperthermia, significantly reducing the risk of toxicity of these drugs in healthy tissues. BRIEF DESCRIPTION OF THE FIGURES

[015] Figure 1 shows the Fourier transform infrared spectra: (a) terpolymer (PLDLA-co-TMC), (b) copolymer (PEO-PPO-PEO), (c) empty nanoparticle, without magnetite.

[016] Figure 2 shows the Fourier transform infrared spectra: a) empty nanoparticle, without magnetite; b) pure magnetite; c) magnetite + hydroxymethylammonium (TMAOH) (Fe3O4#TMAOH); d) magnetite 1-0.25; e) magnetite 1-0.5; f) magnetite 11.0.

[017] Figure 3 shows the diffractograms of the samples: (a) pure magnetite, (b) magnetite + TMAOH (Fe3O4#TMAOH), (c) magnetite 1-0.25; (d) magnetite 1-0.5; (e) magnetite 1-1.0; and (f) pure magnetite (catalog sheet of the diffractogram, ICDD # 19-629).

[018] Figure 4 shows the hysteresis of the samples: a) pure magnetite, b) magnetite 1-0.25; c) magnetite 1-0.5; d) magnetite 1-1.0.

[019] Figure 5 shows the Mossbauer spectrum at 298 K for the samples: a) magnetite; b) magnetite 1-0.25; c) magnetite 1-0.5; d) magnetite 1-1.0.

[020] Figure 6 shows the Fourier transform infrared spectroscopy spectra of (a) the terpolymer (PLDLA-coTMC), (b) the block copolymer (PEO-PPO-PEO), (c) the empty (or blank) PLDLA-co-TMC / PEO-PPO-PEO based nanoparticle, (d) the finasteride-laden nanoparticle and (e) the oral drug finasteride. Petition 870190047245, dated 05 / 20 / 2019, page 19 / 44 6 / 20

[021] Figure 7 shows the thermogravimetric analysis curves of (a) PLDLA-co-TMC terpolymer, (b) PEO-PPO-PEO block copolymer, (c) PLDLA-co-TMC / PEOPPO-PEO based empty (or blank) nanoparticle, (d) finasteride oral drug, and (e) finasteride-laden nanoparticle.

[022] Figure 8 shows the differential scanning calorimetry thermograms of (a) PLDLA-co-TMC terpolymer, (b) finasteride drug, (c) PEO-PPO-PEO block copolymer, (d) PLDLA-co-TMC / PEO-PPO-PEO empty (or blank) nanoparticle, and (e) finasteride-laden nanoparticle.

[023] Figure 9 is a representation of field emission scanning electron microscopy micrographs of (a) the empty (or blank) PLDLA-co-TMC / PEO-PPO-PEO-based nanoparticle and (b) the finasteride-laden nanoparticle.

[024] Figure 10 is a graphical representation of the controlled in vitro release of finasteride-laden nanoparticles. DETAILED DESCRIPTION OF THE INVENTION

[025] The present technology relates to hybrid nanocomposites based on PLDLA-co-TMC@PEO-PPO-PEO and ferrofluid, which can be used for magnetic hyperthermia and drug delivery. The technology also relates to the process of obtaining the hybrid nanocomposites, to the pharmaceutical compositions containing the hybrid nanocomposites and to the use of the hybrid nanocomposites or compositions as drug delivery systems, especially antitumor drugs.

[026] The hybrid nanocomposites of the present technology comprise poly(L-co-D,L lactic acid-co-trimethylene carbonate), abbreviated as PLDLAco-TMC; the block copolymer poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), abbreviated as PEO-PPO-PEO; magnetic nanoparticles, tetramethylammonium hydroxide and a lipo- or hydrophilic drug, preferably an antitumor drug. The nanoparticles Petition 870190047245, dated 05 / 20 / 2019, page 20 / 44 7 / 20 magnetic plates are preferably coated with a polymeric matrix of poly(L-co-D,L lactic acid-co-trimethylene carbonate)@poly(ethylene oxide)poly(propylene oxide)-poly(ethylene oxide), whose abbreviation is PLDLA-coTMC@PEO-PPO-PEO.

[027] Magnetic nanoparticles can be ferrofluid, preferably magnetite (Fe3O4) or maghemite (Fe2O3). For every 1.0 mL of PLDLA-co-TMC@PEO-PPO-PEO there can be 0.2 to 1.2 mL of a dilute suspension of the ferrofluid.

[028] The process for obtaining the hybrid nanocomposites defined above comprises the following steps: a) To synthesize iron oxide nanoparticles in an aqueous medium; b) Prepare an aqueous solution of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO); c) Prepare an organic solution of poly(L-co-D,L lactic acid-cotrimethylene carbonate) (PLDLA-co-TMC); d) Add the diluted suspension prepared in item “a” to the aqueous solution prepared in “b” and stir”, which can be done in an ultrasonic bath; e) Add, while stirring, the organic solution prepared in “c” to the aqueous phase obtained in “d”, the stirring may be done in an ultrasonic bath; f) Remove the organic solvent, which can be done using a rotary evaporator; g) Purify the resulting nanocomposites.

[029] In step “a”, the iron oxide nanoparticles can be Fe3O4 or γFe2O3, and the synthesis can occur by coprecipitation in aqueous medium or in microemulsion, by decomposition of coordination compounds, by sonication, or by reduction of metal ions. Also in “a”, the magnetic fluids obtained can be ionic or surfacted. Petition 870190047245, dated 05 / 20 / 2019, page 21 / 44 8 / 20

[030] In “b”, the PEO-PPO-PEO ratio can be between 0.5 and 25 mg / mL of water. Also in “b”, dissolution can occur with the aid of agitation, preferably in an ultrasonic bath, for 1 to 35 minutes, at a temperature between -5 and 55 °C.

[031] In “c”, the PLDLA-co-TMC ratio can be between 0.25 and 200 mg / mL of organic phase, and the organic phase can be composed of acetone:methanol in proportions between 99:1 and 50:50. Dissolution can occur with the aid of agitation, preferably in an ultrasonic bath, for 1 to 35 minutes, at a temperature between -5 and 55 °C.

[032] In “d” agitation can be for 1 to 35 minutes, preferably in an ultrasonic bath, and the temperature can be maintained between -4oC and 55oC.

[033] In “g”, purification preferably occurs by centrifuging the nanoparticles for 4 minutes to 4 hours and between 10,000 and 25,000 rpm, followed by washing the nanoparticles between two and fifteen times with ultrapure water to remove free magnetite or excess polymers. The present technology also refers to pharmaceutical compositions containing the hybrid nanocomposite defined above, pharmaceutically and pharmacologically acceptable drug and excipients.

[034] The hybrid nanocomposites defined above, as well as pharmaceutical compositions, can be used for drug manufacturing, preferably antitumor drugs.

[035] The present technology can be better understood in accordance with the following non-limiting examples. Example 1: Synthesis of nanocomposites - Fe3O4#TMAOH@PLDLA-COTMC@PEO-PPO-PEO

[036] Nanocomposites were synthesized by mixing predefined amounts of the terpolymer PLDLA-co-TMC with the copolymer PEOPPO-PEO, forming the empty nanoparticle without magnetite. When 0.25, 0.5, or 1.0 were added to PLDLA-co-TMC and PEO-PPO-PEO Petition 870190047245, dated 05 / 20 / 2019, page 22 / 44 9 / 20 mL of a diluted suspension of the ferrofluid Fe3O4#TMAOH, the samples of the hybrid nanocomposites (FeXOY#TMAOH@PLDLA-co-TMC@PEOPPO-PEO) were named mag1-0.25; mag1-0.5; and mag1-1.0, respectively.

[037] For the synthesis of hybrid nanocomposites (Fe3O4#TMAOH@PLDLA-CO-TMC@PEO-PPO-PEO), initially the Fe3O4 nanoparticles functionalized with TMAOH were obtained by the reduction-precipitation method. For this, 15 mL of 1 mol L-1 Na2SO3 was mixed with 22.5 mL of 2 mol L-1 FeCl3.6H2O previously dissolved in 0.5 mol L-1 HCl. The mixture was carried out in a 1000 mL round-bottom flask while bubbling N2 gas. After mixing Fe3+ and (SO3)2, the solution's color changed from light yellow to red and back to yellow. Then, 600 mL of a 0.5 mol L-1 ammonium hydroxide solution were poured into the initial solution, under vigorous stirring, forming a black precipitate. After 30 minutes, the suspension was then centrifuged at 3000 rpm for 2 minutes. In order to functionalize the magnetite, 6 mL of 25% TMAOH were added to the product and 2 mL of this suspension were diluted ten times in distilled water to obtain the ferrofluid used in coating the polymer.

[038] After the nanoparticles were synthesized, functionalized, and diluted, magnetite was incorporated into the polymer following these steps:

[039] Step 1: Synthesis of polymeric nanoparticles based on poly(L-coD,L lactic acid-co-trimethylene carbonate) (PLDLA-co-TMC)@poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) by the solvent displacement method. Dissolve between 5 and 100 mg of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) in 4 to 150 mL of Milli-Q water using an ultrasonic bath for 1 to 35 minutes, at a temperature between -5 and 55 °C. In parallel, dissolve between 5 and 200 mg of poly(L-co-D,L lactic acid) Petition 870190047245, dated 05 / 20 / 2019, page 23 / 44 Dissolve 10 / 20 co-trimethylene carbonate (PLDLA-co-TMC) in a volume between 1 and 20 mL of organic phase (acetone:methanol ratio between 99:1 and 50:50) using an ultrasonic bath for 1 to 35 minutes, at a temperature between -5 and 55 °C for 1 to 35 minutes. Add between 0.5 and 2.0 mg of finasteride and shake the solution.

[040] Step 2: In the samples containing the magnetic ferrofluid, volumes between 0.01 mL and 3 mL of functionalized and diluted suspension of the magnetic fluid previously prepared as described above (ferrofluid) were added to the aqueous phase described above. The suspension was stirred for between 1 minute and 35 minutes using an ultrasonic bath. The temperature was maintained between -4°C and 55°C.

[041] Step 3: the organic phase described above was added, drop by drop, to the aqueous phase, under agitation, in an ultrasonic bath.

[042] Step 4: the organic solvent was removed with a rotary evaporator, but any other equipment that allows this removal can be used.

[043] When the nanoparticles contained a drug, it was added to the organic phase described above. After solvent removal, the nanoparticles were centrifuged for 4 minutes to 4 hours at speeds between 10,000 rpm and 25,000 rpm.

[044] The nanoparticles were washed between two and fifteen times with Milli-Q water to remove free magnetite or excess polymers.

[045] Once the hybrid nanocomposites were centrifuged, the precipitate was sent for analysis by Fourier Transform Infrared Spectroscopy (FTIR), X-ray diffraction (XRD), Mossbauer spectroscopy and magnetization measurements by Vibrating Sample Magnetometry (VSM). The resuspended sediment was frozen in liquid nitrogen, lyophilized (L101, Liotop) and kept at -70 °C. Example 2 - Characterization of nanocomposites Petition 870190047245, dated 05 / 20 / 2019, page 24 / 44 11 / 20

[046] Observing Figure 1, which presents the Fourier transform infrared spectra: (a) terpolymer (PLDLA-coTMC), (b) copolymer (PEO-PPO-PEO), (c) empty nanoparticle, without magnetite, characteristic bands can be verified. In Figure 1(a), the presence of the characteristic bands of PLDLA-co-TMC (1753, 1455, 1380, 1266, 1187, 1091, 792 and 753 cm-1) is noted, attributed to the C=O, CH3, CH2, =CO, COC, CH and -CH2CH2 vibrations of trimethylene carbonate (TMC), respectively. In Figure 1(b), PEO-PPO-PEO was identified by the bands 1343 and 1110 cm-1, attributed to the OH and CO vibrations, respectively. The junction of PLDLA-co-TMC with PEO-PPOPEO was confirmed by the peaks 1753 and 1110 cm-1, attributed to the C=O (PLDLA-co-TMC) and CO (PEO-PPO-PEO) vibrations, respectively (Figure 1 (c)).

[047] Figure 2 shows the FTIR spectra of: a) empty nanoparticle, without magnetite, b) pure magnetite, c) magnetite + TMAOH (Fe3O4#TMAOH), d) mag1-0.25; e) mag1-0.5; ef) mag1-1.0. Figures 2 (b) and (c) show the presence of Fe3O4, identified by the 581 cm-1 band (Fe-O) and, in the ferrofluid, Fe3O4-TMAOH, by the 977 cm-1 band (CN), respectively. Analyzing Figures 2 (d), (e) and (f), the coating of the ferrofluid Fe3O4-TMAOH by the PLDLA-coTMC@PEO-PPO-PEO matrix is ​​observed, through the presence of the bands at 580 cm-1, of Fe3O4, attributed to the Fe-O bond, and the presence of the polymer bands. This suggests that the magnetic nanoparticles were coated, demonstrating the efficiency of the solvent displacement method in coating them.Furthermore, some bands associated with CO from the PLDLA-co-TMC sample (1116 cm⁻¹ and 1280 cm⁻¹) were displaced in the hybrid nanocomposites (1110 cm⁻¹ and 1255 cm⁻¹), suggesting an intermolecular interaction between Fe₃O₄-TMAOH (-O and N) and PLDLA-co-TMC (CO). It can be observed that, as the amount of magnetite added to the polymer increased (0.25 < 0.50 < 1.0), the intensity of... Petition 870190047245, dated 05 / 20 / 2019, page 25 / 44 The band related to the Fe-O bond increased in the 12 / 20 band, while the signals associated with PLDLA-co-TMC decreased at 842 cm⁻¹(CH), 792 cm⁻¹(CH), and 753 cm⁻¹(-CH₂CH₂-). Considering that the mag1-1.0 sample has the lowest coating mass and highest magnetite concentration, the absence of the signal may suggest that the number of CH bonds available in PLDLA-co-TMC was reduced, compensating for the greater number of Fe₃O₄-TMAOH bonds. Therefore, Figure 2 confirms the efficiency of the solvent displacement method for obtaining nanocomposites based on PLDLA-co-TMC@PEO-PPO-PEO@Fe₃O₄#TMAOH. That is, PLDLA-co-TMC successfully coated the magnetic nanoparticles, generating hybrid nanocomposites.

[048] Observing Figure 3, which presents the XRD of the samples: (a) pure magnetite, (b) magnetite + TMAOH (Fe3O4#TMAOH), (c) mag1-0.25; (d) mag1-0.5; (e) mag1-1.0; and (f) pure magnetite, it can be seen that the synthesized magnetite nanoparticles exhibit a crystalline structure with intense peaks at 2Θ = 30.0°, 35.4°, 43.0°, 53.4°, 56.9° and 62.5°, corresponding to the diffraction of the magnetite crystal planes 220, 311, 400, 422, 511 and 440, respectively (ICDD # 19-629). The XRD data for the magnetic nanocomposites exhibit the same peaks as magnetite. The peak reflection intensities in these X-ray diffraction patterns for the mag1-0.25 sample are generally lower than those for the mag1-0.5 sample, which in turn are lower than those for the mag1-1.0 sample. As discussed in the FTIR section, this occurs because the mag1-0.25 sample contains a smaller amount of magnetite than the mag1-0.5 sample, which in turn has a smaller amount than the mag1-1.0 sample.It should be noted that no reflection peak was observed for any other crystalline phase; therefore, the only iron oxide was magnetite.

[049] Figure 4 shows the magnetic hysteresis of the samples: a) pure magnetite, b) mag1-0.25; c) mag1-0.5; d) mag1-1.0. The coating Petition 870190047245, dated 05 / 20 / 2019, page 26 / 44 The 13 / 20 difference in the polymer matrix in the hybrid nanoparticles is evident, as the saturation magnetization, Ms, of the hybrid nanocomposites is lower than that of the magnetite sample. Ms depends on the volume fraction of Fe3O4 nanoparticles and the nature of the interactions within the polymer matrix (PLDLA-co-TMC + PEO-PPO-PEO). The remanent magnetization (Mr) of the magnetite was close to zero, suggesting that there was almost no remanent magnetization when the external magnetic field was removed. The magnetic parameters, coercive force and magnetic remanence, demonstrated that the magnetite-based magnetic nanoparticle exhibited superparamagnetism, favoring its redispersion after the removal of the external magnetic field. According to the average diameter of the magnetite-based magnetic nanoparticles (~10 nm), determined by scanning electron microscopy for this sample, each particle corresponds to a single crystalline domain, exhibiting only one orientation of the magnetic field.Therefore, this explains the superparamagnetic property of the magnetic sample. In hybrid nanocomposites, due to sizes being larger than 10 nm, there is no superparamagnetic characteristic. Thus, analyzing Table 1, it can be seen that the saturation magnetization and remanent magnetization values ​​increase with the amount of Fe3O4. This happens due to the strong interaction between nanoparticles within the polymer matrix (Jayakrishnan, P., Ramesan, MT: Studies on the effect of magnetite nanoparticles on magnetic, mechanical, thermal, temperature dependent electrical resistivity and DC conductivity modeling of poly (vinyl alcohol-co-acrylic acid) / Fe3O4 nanocomposites. Mater. Chem. Phys. 186, 513-522 (2017)). Table 1. Magnetic parameters of nanocomposites with different magnetite nanoparticle contents. Sample Ms / my g-1Hc / Oe Mr / my g-1 Magnetite 67 5 0.01 Petition 870190047245, dated 05 / 20 / 2019, page 27 / 44 14 / 20 Nanocomposite (0.25 mL of magnetic fluid) 1.0 58 0.10 Nanocomposite (0.5 mL of magnetic fluid) 1.7 60 0.16 Nanocomposite (1 mL of magnetic fluid) 9.0 60 0.9

[050] Figure 5 shows the 298 K Mossbauer spectrum for the samples: magnetite, mag1-0.25; mag1-0.5; and mag1-1.0. The 298 K Mossbauer spectrum for the magnetite sample (Fig. 5; corresponding hyperfine parameters in Table 2) consists of two sextets, one assigned to high-spin Fe3+ at tetrahedral sites (Bhf = 48.04(3) T) and the other to mixed valence Fe3+ / 2+ at octahedral sites (Bhf = 44.53(5) T) of the magnetite structure. Electron delocalization causes the nucleus to experience an average valence of Fe3+ / 2+. Given that the non-recoiling fraction of octahedral sites at room temperature is 6% smaller than that of tetrahedral sites, the relative area ratio RAoct / RAtet = 1.67 indicates that the magnetite obtained (magnetite sample) is relatively close to its pure stoichiometric form.

[051] From Mossbauer's data regarding the hybrid nanocomposite, we can observe decreasing values ​​for the relative area ratio RAoct / RAtet, 1.53 for the mag1-0.25 sample; 1.47 for the mag1-0.5 sample; and 1.40 for the mag1-1.0 sample. This decrease indicates that Fe2+ is being oxidized to Fe3+. These results suggest that increasing the amount of Fe3O4 increases the difficulty of encapsulating Fe3O4. It is known that polymeric coatings promote the chemical protection of magnetite, increasing stability and preventing it from being degraded during or after the preparation of the hybrid nanocomposite. Thus, these results suggest that the Petition 870190047245, dated 05 / 20 / 2019, page 28 / 44 15 / 20 The amount of magnetite added to the mag1-1.0 sample was greater than the amount of polymer used. Therefore, the magnetite not incorporated into the polymer in this sample was partially oxidized. Table 2. Assembly parameters of Mossbauer57Fe spectra recorded at 298 K. δ = isomeric shift relative to aFe; 2ε = quadrupole shift; Γ = resonance line width; Bhf = hyperfine magnetic field; RA = relative subspectral area. Denotes tetrahedral [ ] and octahedral {} symmetries of the Fe-O coordination sites of the spinel structure. [Fe3+] and {Fe3+ / 2+} stand for iron in tetrahedral and octahedral coordination sites of the magnetite spinel structure, respectively. The numbers in parentheses are the standard deviations about the last significant digit of the value, as output by the computer fitting program, based on the least squares algorithm. Sample 57Fe site ---r / ____-Γ δ / mm s „ -_____-1 2e / mm s Γ / mm s-1 Bh / T RA / % Magnetite Fe3O4 [Fe3+] 0.314(5) -0.009(9) 0.47(2) 48.04(3) 37.4(1) {Fe3+ / 2+} 0.569(8) 0.00(1) 0.87(3) 44.53(5) 62.6(1) Nanocomposite (0.25 mL Fe3O4 [Fe3+] 0.31(1) -0.01(2) 0.51(5) 47.89(8) 39.6(1) ferrofluid) {Fe3+ / 2+} 0.55(2) -0.04(3) 0.88(6) 44.5(1) 60.4(1) Nanocomposite (0.5 mL Fe3O4 [Fe3+] 0.33(1) 0.00(3) 0.50(4) 47.97(9) 40.5(1) ferrofluid) {Fe3+ / 2+} 0.57(2) 0.01(3) 0.81(6) 44.2(1) 59.5(1) Nanocomposite (1 mL Fe3O4 [Fe3+] 0.323(3) -0.005(7) 0.59(2) 48.14(3) 41.7(1) ferrofluid) {Fe3+ / 2+} 0.498(9) 0.01(2) 1.22(3) 43.8(1) 58.3(1) Example 3 - Preparation and characterization of PLDLA-co-TMC and PEO-PPO-PEO based polymeric carriers containing a lipophilic drug. Petition 870190047245, dated 05 / 20 / 2019, page 29 / 44 16 / 20

[052] The production process of PLDLA-co-TMC / PEO-PPO-PEO nanoparticles for the controlled release of lipophilic drugs comprises the following steps: a) Dissolve between 10 and 60 mg of PEO-PPO-PEO in 5 to 100 mL of Milli-Q water using an ultrasonic bath for 1 to 30 minutes at a temperature of -5 to +50 °C. Then, dissolve between 10 and 50 mg of PLDLA-co-TMC in a volume between 1.0 and 10.0 mL of organic phase (acetone:methanol ratio between 99:1 and 50:50) using an ultrasonic bath for 1 to 30 minutes at a temperature of -5 to +50 °C. b) Add a lipid-soluble drug to the organic phase. Stir for 1 to 30 minutes using an ultrasonic bath. The temperature can be maintained between -5 and 50 °C. c) Add the organic phase to the aqueous phase drop by drop or rapidly, while stirring in an ultrasonic bath. d) Remove the organic solvent using a rotary evaporator, or any other equipment that allows for this removal. e) Centrifuge the nanoparticles loaded with a lipophilic drug for 5 minutes to 2 hours at 10,000 to 20,000 rpm. f) Wash the nanoparticles between two and ten times with MiliQ water to remove free drug or excess polymers.

[053] Observing Figure 6 (a) it can be verified that the PLDLA-co-TMC terpolymer presented the characteristic bands at 1753, 1450, 1380, 1266, 1185, 792 and 753 cm⁻¹ which are attributed to the C=O, CH₃, CH₂, =CO, COC, CH and -CH₂CH₂ vibrations of trimethylene carbonate (TMC), respectively. Considering Figure 6(b), PEO-PPO-PEO was identified by the bands 1343 and 1110 cm⁻¹ which were attributed to the OH and CO vibrations, respectively. The combination of PLDLA-coPetition 870190047245, of 20 / 05 / 2019, p. 30 / 44 17 / 20 The TMC with PEO-PPO-PEO was demonstrated by the peaks at 1753 and 1110 cm⁻¹ attributed to the C=O (PLDLA-co-TMC) and CO (PEO-PPO-PEO) vibrations, respectively (Figure 6(c)). Analyzing Figures 6(d) and (e), the encapsulation of finasteride by the PLDLA-co-TMC / PEO-PPO-PEO matrix is ​​observed, Figure 6(d), which confirmed the presence of finasteride through the 1668 cm⁻¹ band attributed to the -C=O bond. Therefore, Figure 6 proves the efficiency of the solvent displacement method for processing PLDLA-co-TMC / PEO-PPO-PEO-based nanoparticles and its ability to encapsulate finasteride.

[054] Figure 7 (a), (b) and (d) confirms a degradation step for PLDLA-co-TMC (97.6% -280 °C to 370 °C), PEO-PPO-PEO (97.1% -215 °C to 415 °C) and finasteride (93.7% -264.2 °C to 387.3 °C). The mass loss of finasteride is due to amide, ketone and alkyl groups. It is possible to see that Figure 7 (c) shows two stages of mass loss associated with a loss of 39.0% below 314 °C (PLDLA-co-TMC) and a loss of 54% between 314 °C and 409 °C (PEO-PPO-PEO), respectively. This result confirms the obtaining of nanoparticles based on PLDLA-co-TMC / PEO-PPO-PEO. It is also noted that finasteride-laden nanoparticles exhibit two stages of decomposition, Figure 7 (e). The initial and final temperatures of the PEO-PPO-PEO region are lower when compared to the same initial and final decomposition temperatures of PEO-PPO-PEO Figure 7 (c).Furthermore, Figure 7 (e) indicates that the growth in the percentage of residual carbon suggests that finasteride is encapsulated in PLDLA-co-MTC / PEO-PPO-PEO based nanoparticles.

[055] Figure 8 (a) shows that PLDLA-co-TMC exhibits a glass transition temperature of 50 °C associated with the insertion of trimethylene carbonate into PLDLA. In Figure 8 (b) a characteristic peak is observed at 258.9 °C equivalent to the endothermic melting of finasteride. However, the crystallinity of finasteride was not identified. Petition 870190047245, dated 05 / 20 / 2019, pages 31 / 44 18 / 20 in the finasteride-laden nanoparticle (Figure 8(e)). This result may indicate inhibition of drug recrystallization due to finasteride dispersion in the amorphous form. In Figure 8(c), the single endothermic peak at 54.6 °C corresponds to the melting point and crystallinity of PEO-PPOPEO. In Figure 8(d), a blank (or white) PDLA-co-TMC / PEO-PPO-PEO nanoparticle shows a change in the surfactant-associated melting peak to 53.3 °C. This may suggest an increase in the free volume of PEO-PPO-PEO, displacing the polymer chains and decreasing the melting temperature.

[056] It can be observed that Figure 9 (a) shows the spherical shape, absence of agglomeration and monodispersity of both lyophilized nanoparticles. The diameters of the nanoparticles obtained by electron microscopy are equivalent to the nanoparticle sizes by DLS (Table 3). It is observed that the nanoparticles carrying finasteride showed a possible reduction in diameter. This may be attributed to the sample preparation or the randomness of the sample drop analyzed.

[057] Table 3. Physicochemical characteristics of the nanoparticles. * Formulations (F); Blank nanoparticle (B-NP); Finasteride-loaded nanoparticle (F-NP); mean ± standard deviation (n = 3); finasteride load (FL); encapsulation efficiency (EE); Yield (Y). F B-NP F-NP Z-Ave (nm) 210.0 ±5 256.0 ± 4 Pdl 0.2 ±0.06 0.1 ± 0.01 ZP (mV) -17.0 ±0.3 -19.0 ±0.4 pH (n=3) 5.5 ± 1 5.3 ±1 FL (%) - 3.9 ±0.2 EE (%) - 96.0 ± 2 Y (%) 71.10 ±0.03 55.10 ±0.06

[058] Analyzing Table 3, the solvent displacement method allows obtaining blank nanoparticles (210.0 ± 5 nm, Petition 870190047245, dated 05 / 20 / 2019, pages 32 / 44 19 / 20 suspension concentration 5.6 ± 0.4 mg / mL, recovered mass of 42 ± 3 mg, yielded 71.10 ± 0.03%) and finasteride-laden nanoparticles (256.0 ± 4 nm, suspension concentration 4.30 ± 0.05 mg / mL, recovered mass of 32.4 ± 4.4 mg, yield of 55.10 ± 0.06%) in biomedical ranges and viable yields. The narrow polydispersity (PdI < 0.2) suggests a unimodal distribution and a narrow distribution of the total diameter of the nanoparticles. The zeta potential originates from the carboxyl groups of the polyesters (PLDLA-co-TMC) and the ethylene-propylene oxide copolymers (PEO-PPO-PEO). The zeta potential of finasteride-laden nanoparticles showed lower values ​​(-19.0 ± 40.4) and pH (5.3 ± 1) when compared to blank nanoparticles (-17.0 ± 0.3, pH 5.5 ± 1). These changes may be associated with selective adsorption of hydroxyl groups on the nanoparticle surfaces and the acidity of finasteride.This acidity promotes ionization, decreasing the pH of the samples and reducing the zeta potential. However, there was no significant pH disparity in either nanoparticle (Table 1). This indicates that the encapsulation of finasteride does not influence the zeta potential of the nanoparticles.

[059] Figure 10 shows the controlled in vitro release of finasteride from drug-carrying nanoparticles. Finasteride-carrying nanoparticles (6.67 μg mL-1 suspension concentration) resulted in loading and encapsulation efficiencies of approximately 3.9 ± 0.2% and 96.0 ± 2%, respectively. The finasteride loading relative to the mass of recovered nanoparticles (32.4 ± 0.4 mg), or lyophilized mass, was 3.9% (1.25 mg) finasteride equivalent. In terms of encapsulation efficiency, considering 1.3 mg of the theoretical initial mass of finasteride, 96.0 ± 2% (1.25 mg) were encapsulated by the finasteride-carrying nanoparticle, and 3.9 ± 0.2% (0.05 mg) of the drug remained free (Table 3). This high encapsulation efficiency may be associated with a possible polymer matrix / finasteride interaction. Observing Figure 10, the in vitro release of the carried nanoparticles Petition 870190047245, dated 05 / 20 / 2019, pages 33 / 44 A 20 / 20 dose of finasteride demonstrated the release of approximately 50% of the finasteride within the first 24 hours, without the presence of a burst effect, as the release occurs at a constant, controlled rate, up to approximately 4 hours. This is the period during which the finasteride-laden nanoparticles would be circulating in the human body.

Claims

1 - Hybrid nanocomposites, characterized by comprising poly(L-co-D,L lactic acid-co-trimethylene carbonate), abbreviated as PLDLAco-TMC; the block copolymer poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), abbreviated as PEO-PPO-PEO; magnetic nanoparticles, tetramethylammonium hydroxide and a lipo- or hydrophilic drug. 2 - Hybrid nanocomposites, according to claim 1, characterized in that the lipophilic or hydrophilic drug is preferably an antitumor agent. 3 - Hybrid nanocomposites, according to claim 1, characterized in that the magnetic nanoparticles are coated with a polymeric matrix of poly(L-co-D,L lactic acid-co-trimethylene carbonate)@poly(ethylene oxide) - poly(propylene oxide) - poly(ethylene oxide), whose abbreviation is PLDLA-co-TMC@PEO-PPO-PEO. 4 - Hybrid nanocomposites, according to claims 1 and 3, characterized in that the magnetic nanoparticles are ferrofluid, preferably magnetite (Fe3O4) or maghemite (Fe2O3). 5 - Hybrid nanocomposites, according to claims 1 to 4, characterized by containing, for each 1.0 mL of PLDLA-coTMC@PEO-PPO-PEO, 0.2 to 1.2 mL of a diluted suspension of ferrofluid. 6 - Process for obtaining the hybrid nanocomposites defined in claims 1 to 5, characterized by comprising the following steps: a) Synthesizing iron oxide nanoparticles in aqueous medium; b) Preparing an aqueous solution of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO); Petition 870190047245, dated 05 / 20 / 2019, page 35 / 44 2 / 3 c) Preparing an organic solution of poly(L-co-D,L lactic acid-cotrimethylene carbonate) (PLDLA-co-TMC); d) Add the diluted suspension prepared in item “a” to the aqueous solution prepared in “b” and stir”, which can be done in an ultrasonic bath; e) Add, while stirring, the organic solution prepared in “c” to the aqueous phase obtained in “d”, which can be done in an ultrasonic bath; f) Remove the organic solvent, which can be done in a rotary evaporator; g) Purify the obtained nanocomposites. 7 - Process for obtaining hybrid nanocomposites, according to claim 6, step “a”, characterized by the iron oxide nanoparticles being Fe3O4 or γFe2O3, and the synthesis occurring by coprecipitation in aqueous medium or in microemulsion, by decomposition of coordination compounds, by sonication, or by reduction of metal ions. 8 - Process for obtaining hybrid nanocomposites, according to claim 6, step “a”, characterized by the magnetic fluids obtained being ionic or surfactants. 9 - Process for obtaining hybrid nanocomposites, according to claim 6, step “b”, characterized by the PEO-PPO-PEO ratio being between 0.5 and 25 mg / mL of water and the dissolution occurring with the aid of agitation, preferably in an ultrasonic bath, for 1 to 35 minutes, at a temperature between -5 and 55 °C. 10 - Process for obtaining hybrid nanocomposites, according to claim 6, step “c”, characterized by the proportion of PLDLA-co-TMC being between 0.25 and 200 mg / mL of organic phase, the organic phase being composed of acetone:methanol in proportions between 99:1 and 50:50; and the dissolution occurring with the aid of agitation, preferably in an ultrasonic bath, for 1 to 35 minutes, under a temperature between -5 and 55 °C. 11 - Process for obtaining hybrid nanocomposites, according to claim 6, step “g”, characterized by the purification occurring by centrifugation of the nanoparticles for 4 minutes to 4 hours and between 10,000 and 25,000 rpm, followed by washing the nanoparticles between two and fifteen times with ultrapure water. 12 - Pharmaceutical compositions characterized by containing the hybrid nanocomposite defined in claims 1 to 5, a drug, and pharmaceutically and pharmacologically acceptable excipients. 13 - Use of the hybrid nanocomposites defined in claims 1 to 5, characterized by being for the manufacture of a drug, preferably an antitumor drug. 14 - Use of the compositions defined in claim 12 characterized by being for the manufacture of a medicine, preferably an antitumor drug.

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