WO2016094991A1 - Procédé d'obtention de nanoparticules de silice pégylées transporteuses d'agences pharmaceutiques hydrophobes, nanoparticules ainsi obtenues et leurs utilisations - Google Patents

Procédé d'obtention de nanoparticules de silice pégylées transporteuses d'agences pharmaceutiques hydrophobes, nanoparticules ainsi obtenues et leurs utilisations Download PDF

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WO2016094991A1
WO2016094991A1 PCT/BR2015/000202 BR2015000202W WO2016094991A1 WO 2016094991 A1 WO2016094991 A1 WO 2016094991A1 BR 2015000202 W BR2015000202 W BR 2015000202W WO 2016094991 A1 WO2016094991 A1 WO 2016094991A1
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nanoparticles
hydrophobic
minutes
process according
peg
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PCT/BR2015/000202
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English (en)
Portuguese (pt)
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Oswaldo Luiz Alves
Leandro Carneiro FONSECA
Amauri Jardim DE PAULA
Diego Stefani Teodoro Martinez
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Universidade Estadual De Campinas - Unicamp
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Priority claimed from BR102014031688-4A external-priority patent/BR102014031688B1/pt
Priority claimed from BR132015030941A external-priority patent/BR132015030941F1/pt
Application filed by Universidade Estadual De Campinas - Unicamp filed Critical Universidade Estadual De Campinas - Unicamp
Publication of WO2016094991A1 publication Critical patent/WO2016094991A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/04—Polysiloxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/04—Polysiloxanes
    • C08G77/06—Preparatory processes

Definitions

  • the present invention falls within the field of nanotechnology comprising the scientific platforms of material chemistry and solid state chemistry with emphasis on the interaction of nanostructures with biosystems, also known as nanobiotechnology.
  • the present invention relates to a process for obtaining pegylated silica nanoparticles carrying hydrophobic drugs, the silica nanoparticles obtained or not by said process and their use in anti-tumor and anti-inflammatory treatments.
  • Cancer is a disease caused by exacerbated cell growth, invading tissues and organs. This disease is derived from external or environmental factors and in approximately 90% of cases external factors are the main causes of the disease. If discovered early, the disease can be remedied by several treatments, among them are chemotherapy, radiation, hormones, among others. In fact, chemotherapy is the most common type of treatment and involves the use of antitumor drugs.
  • Cancer drugs have several side effects when applied to the body, as a result of the low selectivity of the drug that reaches healthy body cells and, consequently, causes high toxicity. In addition, there is a short circulation time in the blood and high 15 000202
  • violacein a violet-colored pigment produced by the bacterium Chromobacterium violaceum found in the Amazon. Studies conducted by researcher Nelson Duran and collaborators, from the State University of Campinas, showed the characteristics of this drug and its high anti-tumor, antiulcerogenic, antiviral, antibacterial and anti-leishmania ability. It is an extremely important drug in many medical applications, which underscores the need to produce molded nanoparticles for drugs with such characteristics.
  • NK911 polymeric micellar nanocarrier
  • the outer surface of this carrier was subjected to polyethylene glycol functionalization to avoid its uptake by the immune system and favors colloidal stability in hydrophilic solvents.
  • Said technology described in the journal resembles the present invention in that it comprises nanoparticles capable of carrying drugs within them and comprises polyethylene glycol (PEG) on the outer surface.
  • PEG polyethylene glycol
  • the present invention differs from the technology described in the article in that it comprises nanostructure functionalized silica, simultaneously with PEG on the outer surface and with hydrophobic organic functions on the inner surface, providing effective drug transport while maintaining it in a stable manner.
  • silica nanoparticles proposed in the present invention have a rigid surface that allows the surface to functionalize internally with drug-interacting hydrophobic organic groups. Additionally, they are distinct nanoparticles in relation to the nanostructure and chemical composition.
  • nanoparticles consisting of acetylated dendritic polymers whose outer surfaces were functionalized with folic acid binders aimed at obtaining nanoparticles capable of specifically targeting tumor cells.
  • Said cell types show strong expression of membrane receptors for folic acid.
  • the insertion of this chemical species on the nanoparticle surface guarantees its specificity to the target cells.
  • the technology described in the article resembles the present invention in that it comprises antitumor drug carrier nanoparticles, however, they do not have PEG on the outer surface, which is important for the biocompatibility of the nanoparticle in the organism, and it does not understand the inner surface. hydrophobic to keep the drug entrapped during intravenous transport.
  • the present invention comprises PEG-functionalized silica nanoparticles on the outer surface, providing biocompatibility in the body, and keeping the drug trapped within the hydrophobic interior until the target cell is reached. Additionally, they treat different nanoparticles in relation to the nanostructure and chemical composition.
  • WO2009038659 relates to organically modified silica nanoparticles with covalently linked photosensitizers applied for photodynamic drug release.
  • the technology described in WO2009038659 resembles the present invention in that it comprises aromatic groups on the inner surface of the nanoparticle.
  • said organic groups are inserted in the middle of a complex molecular structure characterized by photosensitizers, that is, they are part of an organic entity whose function is solely and exclusively linked to the imaging properties of the nanoparticle in tumors.
  • the document does not mention the drug-encapsulating property of the nanoparticle - it has no antitumor potential - and it does not have polyethylene glycol (PEG) groups on its outer surface - biocompatibility problems and short blood circulation time.
  • the present invention comprises hydrophobic groups on the inner surface capable of retaining the drug in the pores during transport in the blood fluid to the reach of the cancer cell when it is released.
  • the nanoparticle comprises PEG groups on the outer surface, which are important for nanoparticle biocompatibility, high colloidal stability essential for locomotion of the nanostructure in the blood (hydrophilic fluid) and long time of blood circulation.
  • US20100255103 relates to silica nanoparticles for biomedical applications.
  • Technology resembles the present invention in that it comprises a silica nanoparticles whose outer surface is coated with PEG and by encapsulating hydrophobic drugs comprising camptothecin.
  • the nanoparticles claimed in the patent have the following technical problems (solved by the present invention): the interior of the patent nanoparticles comprises magnetic cores such as iron oxide and gold; These are inorganic entities occupying a significant internal volume, impairing the ability to incorporate drug; furthermore, the patent does not claim pore volume, of fundamental importance for said drug encapsulation capacity.
  • the nanoparticles proposed in the present invention comprise porous interior hydrophobic groups for retention of the biological active during the intravenous pathway to the target cell range.
  • US20130274226 relates to silica nanoparticles with conjugated agents.
  • the technology comprises silica nanoparticles whose outer surface is coated with PEG functions and by encapsulating hydrophobic drugs in their . inside.
  • the present invention differs from the technology disclosed in US20130274226 in that it comprises nanoparticles whose inner surface comprises hydrophobic groups capable of retaining the drug during intravenous locomotion, an important feature in order to avoid possible losses of the biological active to the blood fluid during the carriage to the target cell.
  • the technology also has the following problems:
  • the technology uses drug-conjugated silane reagents to form covalent bonding of the drug within the nanoparticle.
  • the present invention maintains the drug retained within the hydrophobic interior during carriage as a function of interactions with hydrophobic groups.
  • the present invention has the versatility of encapsulating a range of drugs with hydrophobic chemical characteristics, since only one batch is required to produce silica nanoparticles. Once nanoparticles are obtained, any type of hydrophobic antitumor drug can be incorporated in the shipyard without the need to produce other batches, saving raw material, energy and production time.
  • the proposed nanoparticles simultaneously comprise hydrophilic outer surface due to the presence of polyethylene glycol and hydrophobic inner surface related to the presence of hydrophobic functional groups. Additionally, nanoparticles have as a differential the versatility to incorporate hydrophobic drugs.
  • the proposed process has the differential that it does not use toxic solvents in its stages, encompassing two strategies of functionalization of the outer surface of nanoparticles, as well as understanding mild and specific conditions to produce nanoparticles with the mentioned characteristics.
  • the present invention relates to a process for obtaining pegylated silica nanoparticles carrying hydrophobic drugs, the silica nanoparticles obtained or not by said process and their use in anti-tumor and anti-inflammatory treatments.
  • the process of obtaining drug-bearing pegylated silica nanoparticles comprises the following steps: dissolution of surfactant in ammonia catalyst and alcohol reaction solvent, stirring of the solution, addition of inorganic silicon precursor in hydrophobic silane, addition of solution obtained in the previous step in the solution obtained in the first step, agitation of the obtained solution, addition of inorganic silicon precursor, centrifugation, supernatant disposal and redispersion in ethanol, functionalization of the external surface of the silica nanoparticles, preparation of an ethanolic solution of HCl, addition of nanoparticles to the solution prepared in the previous step, application ultrasound, centrifugation, supernatant disposal and redispersion in ethanol, suspension of nanoparticles obtained in aqueous solution, addition of hydrophobic drug, mixing of the components comprised in the previous step, separation of nanoparticles, obtaining pegylated nanoparticles carrying hydrophobic drugs.
  • Nanoparticles obtained or not by said process comprise polyethylene glycol (PEG) -functioning outer surface, hydrophobic-functionalized porous inner surface, and encapsulated hydrophobic drug.
  • PEG polyethylene glycol
  • the invention also describes the use of the carrier of hydrophobic drugs pegylated nanoparticles in ⁇ antitumor and anti-inflammatory treatments.
  • Figure 1 Molecular structure of PTES (a), TEOS (b) and CTAB (c).
  • Figure 2 Arrangement of phenyl groups in the spaces between CTAB molecules.
  • Figure 3 Representative image of the starting nanoparticle.
  • Figure 4 Representative image of externally functionalized nanoparticle.
  • Figure 6 Representation of nanoparticle production and its functionalization.
  • Figure 7 Summary of the production of the starting nanoparticles.
  • Figure 8 General representation of the functionalization reaction of the starting nanoparticle with PEGio silane.
  • Figure 9 General representation of the functionalization reaction of GPS silane starting nanoparticle and coupling of PEG40 to terminal epoxy function.
  • Figure 10 Summary process of surfactant extraction.
  • Figure 11 Functionalizations performed and their location in the molecular vehicle.
  • Figure 12 Infrared Vibrational Spectra (FTIR) for starting nanoparticles before and after CTAB removal.
  • FIG. 13 Thermogravimetric (TG) curves and their respective differential thermal analysis (DTA) curves of the starting nanoparticles.
  • Figure 14 NPH30-SiOH N2 adsorption-desorption isotherms and NPH30-CTAB-SiOH.
  • Figure 15 Micrograph (TEM) of internally functionalized silica nanoparticles with phenyl groups after CTAB extraction.
  • FIG. 16 Infrared Vibrational Spectra (FTIR) for NPH30-SiO-1 OPEG10.
  • Figure 17 NPH30-SiO-10PEG10 TG and DTA curves.
  • Figure 19 NPH30-SiO-10PEG10 carbon NMR spectrum.
  • Figure 20 NPH30-SiO-10PEG10 silicon NMR spectrum.
  • Figure 21 Infrared Vibrational Spectrum (FTIR) for NPH30-SiO-25PEG10.
  • Figure 22 TG and DTA curves of NPH30-SiO-25PEG10.
  • Figure 23 NPH30-SiO-25PEG10 N2 adsorption-desorption isotherms.
  • Figure 24 NPH30-SiO-25PEG10 carbon NMR spectrum.
  • Figure 26 Infrared Vibrational Spectrum (FTIR) for NPH30-SiO-50PEG10.
  • Figure 30 NPH30-SiO-50PEG10 silicon NMR spectrum.
  • Figure 31 Micrograph (TEM) of silica nanoparticles functionalized internally with phenyl groups and externally with polyethylene glycol.
  • Figure 32 Infrared Vibrational Spectra (FTIR) for NPH30-SIO-10GPS-PEG40.Error! Reference source not found.
  • Figure 33 TG and DTA curves of NPH30-SiO-10GPS-PEG40.
  • Figure 35 13 C NMR spectra of NPH30-SiO-10GPS-PEG40.
  • Figure 36 NPH30-SiO-10GPS silicon NMR spectrum.
  • Figure 37 Colloidal stability graph of nanoparticles at 250 g / mL in PBS.
  • Figure 38 Dose response curves of nanoparticles incubated in red blood cells.
  • Figure 39 Study of nanoparticle aggregation in PBS for one hour.
  • Figure 40 Displays the curcumin calibration curve in ethanol as well as the information for each spectrum acquired at each of the five known concentrations.
  • Figure 41 Graphs the concentration of curcumin in the pegylated silica nanoparticles after each separation by centrifugation and decantation.
  • Figure 42 shows the image of suspensions of silica nanoparticles NPH30-SiOH, NPH30-1 OPEGsoo, NPH30-50PEG 5 and oo-NPH30 l ⁇ PEG5ooo in the presence of curcumin and said drug added to water in the absence of molecular vehicles ( H2O) after 24 hours of decantation and prior to removal of the supernatant.
  • H2O molecular vehicles
  • the present invention comprises the process of obtaining drug-bearing pegylated silica nanoparticles, the steps of which comprise:
  • surfactant dissolution between 0.65 and 0.75 g, preferably 0.75 g, in ammonia catalyst in a concentration between 0.03 and 0.07 mol / L, preferably 0.05 mol / L, and solvent of alcoholic reaction in volumes between 2.5 and 3.8 mL, preferably 3.2 mL;
  • inorganic silicon precursor between 1 and 3 mL, preferably 1.5 mL, in hydrophobic silane;
  • step (c) adding dropwise the solution obtained in step (c) to the solution obtained in step (a);
  • inorganic silicon precursor after 90 minutes of reaction, in volumetric amounts between 100 and 150 ⁇ l, preferably 124 L; f) Centrifugation for 40 to 80 minutes, preferably 60 minutes, between 10,000 and 20,000 rpm, preferably 15,000; discarding the supernatant and redispersing in ethanol;
  • step gl.3 Stirring the solution from step gl.2 between 10 and 20 hours, preferably 13 hours;
  • step gl.4 Centrifugation of the solution comprised in step gl.3 between 40 and 80 minutes, preferably 60 minutes, between 10,000 and 20,000 rpm, preferably 15,000; discarding the supernatant and redispersing in ethanol; repeated at least twice; or
  • g2.1 dispersion of starting nanoparticles in masses between 50 and 500 mg, preferably 300 mg, in ethanol solvent in volumes between 40 and 80 mL, preferably 62 mL and stirring of the solution between 20 and 60 minutes, preferably 30 minutes at room temperature. between 60 and 90 ° C, preferably 85 ° C;
  • g2. Centrifuging the solution between 40 and 80 minutes, preferably 60 minutes, between 10,000 and 20,000 rpm, preferably 15,000 and discarding the supernatant and redispersing in ethanol; repeated at least twice; g2.5) Dispersion of intermediate nanoparticles obtained in step g2. in masses between 50 and 500 mg, preferably 150 mg, in ethanol solvent in volumes between 10 and 30 mL, preferably 20 mL and stirring of the solution between 15 and 45 minutes, preferably 30 minutes at a temperature of 95 ° C;
  • PEG-COOH PEG-terminal carboxylic acid
  • step g2.8 Addition of the PEG-COOH solution from step g2.7 to the solution obtained in step g2.6 for coupling reaction between reactive functional groups of intermediate nanoparticle with reactive functional groups. and PEG-COOH carboxylic acid;
  • step g2.9 Stirring the solution obtained in step g2.8 between 10 and 20 hours, preferably 12 hours at a preferred temperature of 95 ° C; g2.10) Centrifuging the solution obtained in step g2.9 between 40 and 80 minutes, preferably 60 minutes, between 10,000 and 20,000 rpm, preferably 15,000, discarding the supernatant and redispersing in ethanol; repeated at least twice.
  • step (i) Centrifuging the solution obtained in step (i) between 40 and 80 minutes, preferably 60 minutes, between 10,000 and 20,000 rpm, preferably 15,000; discarding the supernatant and redispersing in ethanol; repeated at least twice; and storage until encapsulation of the hydrophobic drug;
  • Suspension of nanoparticles obtained in step (j) in aqueous solution (component 1) is in the range of 5 to 15 mg, preferably 10 mg, and in concentrations up to 1 mg / mL; and adding 0.5 to 2 mg, preferably 1 mg, of hydrophobic drug (component 2); 1) Mixing the components comprised in step (k) by means of ultrasonic homogenization at times between 30 and 60 minutes;
  • the surfactant preferably comprises hexadecyltrimethylammonium bromide
  • the inorganic silicon precursor comprises silicon alkoxides and organosilanes, preferably tetraethylorthosilicate;
  • the hydrophobic silane is used in amounts between 10 and 40 Si-mol%, preferably 30% 'Si-mol;
  • hydrophobic group comprised in hydrophobic silane comprises hydrophobic groups selected from aromatic groups and derivatives; and hydrocarbon groups and derivatives;
  • aromatic groups and derivatives are selected from the group of phenyl, acetophenyl, and pyrene functions;
  • hydrocarbon and derivative groups are selected from the group of octyl and octadecyl functions;
  • - PEG comprised in silane-PEG comprises at least 350 Da; - intermediate silane with reactive functional groups is used in amounts between 2 and 50 mol% Si;
  • reactive functional groups comprised in intermediate silane comprise epoxy functions
  • the strong base used preferably comprises potassium hydroxide
  • the PEG-COOH used comprises at least 350 Da
  • hydrophobic drug is used in amounts from 1 to 20% by mass;
  • - hydrophobic drug used is selected from the group of drugs comprising curcumin ; camptothecin, doxorubicin, violacein, tamoxifen, beta-lapacone and the like, preferably camptothecin.
  • the separation means described in the process comprise centrifugation or decantation.
  • Centrifugation is performed at rotational forces between 1500 and 2000 rpm, preferably for 2 minutes.
  • the decantation is carried out in time of at least 24 hours.
  • sequence of steps k, l, m and n may be performed by the following sequence of steps:
  • step (k) Centrifugation of nanoparticles from step (k) under the same conditions established in step (j) and obtaining pegylated nanoparticles carrying hydrophobic drugs.
  • the pegylated hydrophobic drug carrier nanoparticles of the present invention comprise the following characteristics: outer surface functionalized with polyethylene glycol (PEG); porous inner surface functionalized with hydrophobic groups;
  • pegylated nanoparticles carrying hydrophobic drugs comprise the following characteristics:
  • hydrophobic groups selected from aromatic groups and derivatives; and hydrocarbon groups and derivatives;
  • hydrophobic drug selected from the group of drugs comprising curcumin, camptothecin, doxorubicin, violacein, tamoxifen, beta-lapacone and the like, preferably curcumin and camptothecin;
  • the present invention further comprises the use of pegylated hydrophobic drug carrier nanoparticles in anti-tumor and anti-inflammatory treatments.
  • the process of producing the pegylated hydrophobic drug carrier nanoparticles is initiated with the surfactant, the silane to be internally disposed in the nanoparticle, the inorganic silicon precursor that will form the inorganic wall, and the catalyst.
  • an additional amount of inorganic silicon precursors are implemented by controlling time so that excessive (excess reaction time) or precarious (reaction time) polymerization is avoided as they promote the formation of silicon walls. silica very thick and significantly thin respectively. Therefore, adequate nucleation time control is required.
  • the external surface can be functionalized with the silane of interest, followed by the removal of the surfactant.
  • reaction parameters reaction time and temperature
  • the production process of the hydrophobic drug-bearing pegylated nanoparticles is initiated by the co-condensation process involving the inorganic silicon precursor (TEOS), comprising the inorganic source of silica, hydrophobic silane comprising hydrophobic groups selected from the aromatic and derivative groups (phenyl, acetophenyl and pyrene) and hydrocarbon and derivative groups (octyl and octadecyl) which are located on the inner surface of the silica, the surfactant preferably comprising hexadecyltrimethylammonium bromide (CTAB), the function of which is to form spherical micelles, whereby aqueous ammonium hydroxide as catalyst and ethanol as reaction solvent as a template.
  • CTAB hexadecyltrimethylammonium bromide
  • TEOS and hydrophobic silane mutually hydrolyze and initiate their polymerization around the micelles, so that they are muted. that "silica. negatively charged electrostatically interacts with. . the positively charged micellar surface and hydrophobic groups arrange themselves within the small spaces between the hydrophobic tails of the surfactants. This situation can best be seen in Figure 2.
  • the outer surface is also subjected to a novel process of functionalization (after functionalization), whose goal is to insert bodily functions which confer new 'properties nanoparticles , as observed in Figure A ..
  • functionalization a novel process of functionalization
  • the group ' R indicated in. nanoparticle represents any organic function to be grafted to the outer surface through a previously condensed silane.
  • the main strategy is to produce antagonistic nanoparticles, i.e. covering hydrophobic interior and hydrophilic outer surface due to the applications in which they are expected.
  • the major silanes involved in external surface functionalizations are compounds whose R groups encompass strategic organic functions, such as glycidoxypropyltrimethoxy silane (GPS silane), as well as fundamental functions for the acquisition of important new chemical properties, including 2. -
  • the surfactant is removed by extraction with ethanolic HCl solution and sonication as discussed above.
  • Developed nanoparticles have potential application as carriers of antitumor hydrophobic drugs and, as such, must have the ability to trap a drug within them for later delivery to the cancer cell and colloidal stability in the blood to ensure biological target range.
  • the importance of the presence of internal hydrophobic groups is noteworthy due to their hydrophobic character similar to the antitumor drugs that also have this characteristic, resulting in a strong interaction between Van der Waals and both, favoring their effective insertion. and storage within the nanostructure.
  • PEG hydrophilic
  • polyethylene glycol also contributes to "softening" the protein / nanoparticle interaction, especially those involved in the recognition and withdrawal of supposedly foreign agents in the blood, as occurs in the immune system.
  • FTIR Fourier transform infrared
  • the analysis was performed with a 90 ° single pulse excitation of 5.0 ⁇ in channel 1 ( 29 Si) followed by signal acquisition under a continuous uncoupling pulse in channel 2 ( 1 H), initiated simultaneously with channel 1 pulse. , until the signal relaxation in that channel .
  • 13 C NMR spectra were obtained with the cross-polarization and magic angle spinning (CPMAS) method of the neighboring IH nuclei (20480 scans), acquisition time 0.0499 s, pulse interval 3.0 s and frequencies 75.475 MHz on channel 1 ( 13 C) and 300, 131 MHz on channel 2 (3 ⁇ 4)
  • the analysis is performed with a 90 ° pulse excitation of 2.7 ⁇ on channel 2 ( 1 H), followed by a pulse 4000 ⁇ on channel 1 ( 13 C), followed by signal acquisition Along with channel 1 ( 13 C) pulse, another pulse for channel 2 ( X H) decoupling is produced, which extends to relaxation signal on channel 1.
  • thermogravimetric analyzes were performed on a TA Instruments 500 thermal analyzer, TGA module 2050. Analyzes used approximately 5.0 mg of dry samples placed in a platinum crucible. The heating rate was maintained at 10.0 ° C / min and the air flow at 100 mL / min. The morphology of the nanostructures was analyzed by transmission electron microscopy in bright field mode (TEM, Zeiss Libra 120, operating at 80KV). Dynamic light scattering (DLS) to evaluate particle sizes and zeta potential (PZ) were obtained with the Malvern ZetaSizer-Nano instrument.
  • TEM transmission electron microscopy in bright field mode
  • DLS Dynamic light scattering
  • PZ zeta potential
  • DLS results were obtained using deionized water as dispersant (1.0 mg / mL).
  • BET Brunauer-Emmett-Teller
  • P / Po single-point adsorption amount
  • step A The CTAB-containing nanoparticles and phenyl groups produced in step A were subjected to the functionalization process with short-chain PEG silane, constituting step B described in this topic.
  • 300 mg of the starting nanoparticle is dispersed in 40 mL of absolute ethanol for 60 minutes and then homogenized for a further 15 minutes at 60 ° C.
  • PEGio silane is added based on the number of moles of silicon, ie, considering 300 mg of initial NP, the amount of functionalized organosilane corresponds to 10%, 25% or 50% mol-Si relative to number of moles of total silicon present in said nanoparticle mass.
  • Each reaction was performed under constant stirring at 60 ° C for one hour, and an additional 12 hours of functionalization at room temperature.
  • the products were centrifuged and dispersed in ethanol. Removal of the organic mold was performed according to the procedure described below.
  • GPS silane is the intermediate chemical species for obtaining long-chain PEG-containing nanoparticles. It is important to mention that in this reaction a long-chain PEG silane was not used due to the absence of this product for purchase on the market until mid-May 2013.
  • Production of the surface glycoxy-oxide nanoparticles was performed using 300 mg nanoparticles. synthesized in step A dispersed in 62 mL of absolute ethanol for 30 minutes in the sonicator.
  • GPS silane (10 mol% silicon) is added to the flask containing the nanoparticles already suspended in the solvent, followed by stirring of the system for 12 hours at 85 ° C under nitrogen atmosphere. The products were centrifuged at 15000 rpm for one hour and dispersed in ethanol.
  • Figure 9 indicates the production of the silica nanoparticle holding PEG10 in a summarized manner.
  • the synthetic procedure of said molecular vehicle comprises two main steps characterized, first, by the functionalization of GPS silane, followed by the coupling reaction of PEG40 in the epoxy function (significantly reactive) of the mentioned organosilane.
  • the organic mold is still present, requiring a standard processol for all situations that favor the removal of the surfactant that once directed the shape acquired by the nanocarriers.
  • the standard stoichiometric ratio was 5 mg of products: 1 mL of HCl / Ethanol solution.
  • Figure 10 generally represents the removal process of the starting nanoparticle soft template equally applied to the other nanoparticles obtained in the present invention. Evaluation of colloidal stability of nanoparticles
  • the nanoparticles were subjected to the colloidal stability test in order to evaluate the influence of polyethylene glycol on these dispersion aspects.
  • the colloids were dispersed in PBS (1x) at a concentration of 250 pg / mL and centrifuged at rotations of 0, 94, 2348, 9391 and 18407 rcf - the preparation of PBS is described below.
  • the 1 mL volume of each supernatant was subjected to analysis by UV-Vis absorption electron spectroscopy and the absorbance at 263 nm wavelength measured.
  • the nanoparticles produced were subjected to hemolytic assays.
  • PBS (2x) Two tablets of saline phosphate buffer were diluted in 200 mL of deionized water (2 mM phosphate, 0.54 mM KCl and 27.4 mM NaCl);
  • PBS (1x) 100 mL of the above solution was diluted in 100 mL of deionized water (1 mM phosphate, 0.27 mM KCl and 13.7 mM NaCl).
  • the volume of 10 mL of blood was centrifuged at 14,000 rpm at 4 ° C for 10 minutes. The supernatant was discarded and 20 mL of PBS (2x) was added to the pellet which was subsequently resuspended. After this process, two new washes were performed following the current procedure, obtaining 20 mL of a red blood cell solution in PBS (2x). Of this total, 2.5 mL were diluted in 12.5 mL of PBS (2x) to obtain the red cell stock solution.
  • Nanoparticle pretreatment for hemolytic assays comprises a procedure in which all are centrifuged at 15,000 rpm at 4 ° C for 30 min and dispersed in deionized water. This process is repeated twice more to obtain final colloidal dispersions in deionized water at concentrations of 1 mg / mL.
  • Performing hemolytic assays requires a prior assessment of the aggregation behavior of each nanoparticle in PBS for one hour.
  • 25 pL of each molecular carrier was added, respectively, to a mixture of 25 pL PBS (2x) and 950 pL PBS (1x).
  • the dynamic light scattering technique intermittently evaluated the particle size variation and polydispersity index within one hour, similar to the standard red cell incubation time. Colloidal behavior was evaluated at 0, 5, 15, 30, 45 and 60 minutes.
  • each nanoparticle was added in eppendorf containing a mixture of 25 ⁇ l PBS (2x) and 850 ⁇ l PBS (1x). The nanoparticles were kept in the respective vials for one hour. After this step, 100 pL of red blood cell stock solution was added while maintaining one hour of incubation.
  • N nanoparticle
  • xGPS x% of theoretically functionalized glycidoxy groups
  • NPH30-CTAB-SiOH starting nanoparticle containing 30% theoretically functionalized phenyl groups on the inner surface, further containing the surfactant;
  • NPH30-SiOH starting nanoparticle containing 30% of theoretically functionalized phenyl groups on the inner surface with surfactant already removed
  • NPH30-C AB-SiO-xPEG10 nanoparticles containing 30% of theoretically functionalized phenyl groups in the presence of CTAB and x% of theoretically functionalized PEGio groups
  • NPH30-SiO-xPEGio nanoparticles containing 30% of theoretically functionalized phenyl groups in the absence of CTAB and x% of theoretically functionalized PEGio groups;
  • NPH30-CTAB-SiO-xGPS nanoparticle containing 30% of theoretically functionalized phenyl groups in the presence of CTAB and x% of theoretically functionalized glycidoxy groups;
  • NPH30-SiO-xGPS nanoparticle containing 30% of theoretically functionalized phenyl groups in the absence of CTAB and x% of theoretically functionalized glycidoxy groups
  • NPH30-CTAB-SiO-xGPS-PEG / io nanoparticle containing 30% of theoretically functionalized phenyl groups in the presence of CTAB and x% of theoretically functionalized PEG40-coupled glycidoxy groups
  • NPH30-SiO-xGPS-PEG40 nanoparticle containing 30% of theoretically functionalized phenyl groups in the absence of CTAB and x% of theoretically functionalized PEG40-coupled glycidoxy groups.
  • SiO (representing the "shell" of the nanoparticle), such as PH30-CTAB-, PH30-, refer to the chemical entities present within the nanoparticle whereas names written after the SiO nomenclature, such as xPEGio, xGPS and xGPS-PEG-jo, come from the chemical species present on the outer surface.
  • Figure 11 shows the "shell" of internally functionalized silica with phenyl groups and externally with glycidoxy (GPS), open-ring glycidoxy (Open GPS), PEG40-coupled glycidoxy (GPS-PEG-jo) and PEGio functions.
  • GPS glycidoxy
  • Open GPS open-ring glycidoxy
  • PEGio functions The numbers and terms written in blue and red, respectively, will be the basis for the interpretation of the carbon and silicon NMR spectra presented in the invention.
  • the functionalizations shown in the figure below are not necessarily present in the same nanoparticle, showing only the types of surface modifications possible on the inner surfaces and those occurring only on the outer surface.
  • GPS silane and PEGio silane are not concomitantly present on the same nanoparticle because they encompass different molecular vehicle development contexts.
  • FIG. 12 shows the infrared spectra of the starting nanoparticles before and after removal of the organic mold.
  • the main silica bands are observed at 1072 cm -1 (asymmetric Si-O-Si stretch), 800 cm -1 (symmetrical Si-O-Si stretch) and 962 cm -1 (Si-OH stretch), 464 cm - 1 (Si-O-Si angular deformation). Confirmation of mold extraction can be obtained by observing the absence of typical CTAB bands in the NPH30-SiOH spectrum.
  • the soft template bands can be observed at: 2926 cm -1 (asymmetric CH stretch in CTAB carbon chain CH2 groups), 2854 cm -1 (symmetrical CH stretch) and 1232 cm -1 (CN stretch). that the visualization of the latter band is made difficult by the overlap with the bands from the asymmetrical stretches of the SiC (Si-O-Si) tetrahedral units by approximately 1200 cm -1 and 1130 cm “ 1 .- Analyzing both spectra, it is noticeable the absence of these bands after the removal of the soft template, characterizing a significant parameter for the follow-up of the organic mold extraction reactions presented in the next topics.
  • FIG. 13 shows the thermogravimetric (TG) curves and their respective differential thermal analysis (DTA) curves for the starting nanoparticles before and after removal of the organic mold.
  • the NPH30-SiOH DTA curve exhibits a major event at 577 ° C, suggesting the thermal decomposition of the phenyl groups and a minor one occurring at 308 ° C from residual CTAB release. Disregarding the discrete event, which in this case is not significant in relation to the total percentage of the sample, there is only the decomposition of functionalized aromatic species beginning and ending at 324 ° C and 716 ° C, respectively.
  • Figure 14 presents the results of NPH30-SiOH nitrogen adsorption-desorption analysis as well as NPH3G-CTAB-SiOH.
  • isotherm of this last nanoparticle its profile did not show a significantly defined pattern, resembling only the type IV isotherm according to the IUPAC classification for porous materials.
  • Type IV isotherms are related to mesoporous materials.
  • This phenomenon is possibly linked to the presence of CTAB which, together with the range of possibly interconnected pores, makes gas release difficult, making adsorption a slightly irreversible process, which is expected considering the complexity of said hybrid system.
  • the pore distribution of the starting molecular carrier holding the amphiphilic macromolecule exhibits a wide variety of pores, comprising a material of complex porous nature due mainly to the high content of internally functionalized phenyl groups in the nanoparticle.
  • Such an effect generates increased internal volume due to the arrangement of the aromatic rings between the surfactant tails. Consequently, in the nucleation stage during the nanoparticle production, the inorganic lattice deposited around the CTAB engenders pores of varying sizes, characterizing the desired uneven surface.
  • the pore distribution graph whose calculations are based on the Barret, Joyner and Halenda model (BJH method), the predominance of pores between 4 nm and 10 nm is observed.
  • Mesoporous materials have pore sizes ranging from 2 to 50 nm), which classifies said material as mesoporous.
  • the pore distribution graph according to the BJH method indicates greater pore predominance in a size range between 2 nm and 10 nm, reflecting CTAB extraction.
  • the removal of said organic mold can be confirmed by analyzing the surface area variation calculated by the method proposed by Brunauer, Emmet-Teller (BET method) before and after this process.
  • BET method Brunauer, Emmet-Teller
  • the surface area ranged from 548 m 2 / g to 898 m2 / g, respectively.
  • the total pore volume of both nanoparticles was 0.96 mL / g and 2.2 mL / g, respectively.
  • Figure 15 shows the micrograph of the NPH30-SiOH in which disordered porous arrangements can be observed featuring an irregular topography.
  • the presence of .30% mol-Si of phenyl groups within the nanoparticles provides an increase in the internal cavity, generating this characteristic on the outer surface not yet coated with PEG.
  • the nanoparticles show apparent colloidal stability, a feature that will be discussed quantitatively below with the starting and functionalized nanoparticles.
  • polyethylene glycol According to Figure 10 it is possible to estimate the average colloidal diameter around 65 nm.
  • the infrared vibrational spectra of the NPH30-SiOPLOG shown in Figure 16 show the characteristic bands of silica and aromatic rings in all situations. Also shown in the Figure is the spectrum of PEGio silane used in functionalization. The main polymer bands can be observed at 1080 cm -1 (asymmetric COC stretch, similar to Si-O-Si in silica), 1040 cm -1 (symmetrical COC stretch, observed as a shoulder), 2867 cm -1 (stretch CH in ethylene glycol groups C3 ⁇ 4) and 1460 cm -1 (angular deformation CH in ethylene glycol CH2 groups).
  • NPH30-CTAB-SiOH on the spectrum denoted as P ⁇ C-SiOH
  • organosilane product ' is generated NPH30-CTAB-SiO-10EEGio.
  • PC-lOPEGio the visualization of the polymer bands is difficult due to the overlap with the silica bands (at 1100 cm “ 1 and 962 cm -1 ) and the CTAB (at 2854 cm -1 ).
  • NPH30-SiO-10PEGio DTA curve shows a less evident temperature difference near 300 ° C compared to NPH30-CTAB-SiO-lOPEGio, since the final nanoparticle does not contain CTAB whose degradation already occurs at that temperature, contributing to the decrease in peak intensity observed.
  • the nitrogen adsorption-desorption analyzes shown in Figure 18 indicated a nanoparticle profile similar to the starting nanoparticles, allowing to state that after functionalization the structures did not change pore profile as desired.
  • the isotherm of Nitrogen resembles a type IV isotherm according to the IUPAC classification, ie nanocarriers are mesoporous in nature.
  • the surface area of the starting nanoparticle is 898 m 2 / g. After functionalization, said value undergoes slight increase to 943 m 2 / g, indicating little 'differences.
  • the starting nanocarriers For the total pore volume determined by the BJH method, the starting nanocarriers have 2.2 cmVg as the NPH30-SiO-10PEG 10 holds 1.48 cm 3 / g. This difference is possibly linked to the presence of PEGio silane, possibly characterizing steric effects against nitrogen gas adsorption in the nanoparticle.
  • Q 4 Silicon core bonded with 4 substituents OSi, Si (O-Si) 4, located mainly within the "shell" of the silica;
  • Q 3 refers to the silicon core bonded to 3 O-Si substituents and an OH, HO-Si (O-Si) 3 group present on the inner and outer surfaces of the molecular carrier, as well as within the silica layer - used for confirmation of condensation of silanes;
  • Q 2 although less, it is the silicon atom bonded to 2 O-Si groups and 2 OH, (HO) 2Si (O-Si) 2 groups, arranged spatially in the same way as Q 3 nuclei;
  • T 3 These are silicon cores bonded to 3 O-Si substituents and a carbon substituent contained in the group R, R-Si (O-Si) 3, present on the inner surface, inside or on the shell.
  • molecular carrier T 2 : silicon nuclei attached to 2 O-Si substituents, one OH group and one carbon substituent contained in one group, R-Si (0-Si) 20H, present on the inner surface, inside or on the surface.
  • "shell" molecular vehicle silicon nuclei attached to 2 O-Si substituents, one OH group and one carbon substituent contained in one group, R-Si (0-Si) 20H, present on the inner surface, inside or on the surface.
  • R refers to the phenyl, glycidoxy or PEG10 groups present in the nanoparticles.
  • Figure 25 shows NMR spectra of the NPH30-SiO-1OPEGio sample, before and after functionalization, containing the major silicon sites and their corresponding area in percent.
  • the chemical displacements of the silicon peaks have specific values and do not change after functionalization, since the chemical entity that represents them does not undergo structural changes. In fact, what is observed are variations in peak area before and after grafting. Silicon NMR peaks at -110 ppm, -100 ppm and -90 ppm are clear for Q 4 , Q 3 and Q 2 sites , respectively, -80 ppm and -70 ppm from T 3 and T 2 sites at this time. order.
  • the OH groups from the Q 3 and Q 2 sites undergo nucleophilic substitution with ossilanes, generating new T 3 and T 2 sites .
  • NPH30-SiO-25PEG10 The nanoparticle containing internal phenyl (30% mol-Si) groups as well as externally functionalized 25% mol-Si PEG10 groups was called NPH30-SiO-25PEG10. Their respective structural characterizations showed satisfactory results according to the pre-established expectations for the construction of the said molecular vehicle.
  • PC-SiOH subtitles, PC-P and 25PEGio 25PEGio refer respectively to NPH30-SiOH, NHPH30 CTAB--SiO-SiO-25PEGio and NPH30 25PEGi-0.
  • Figure 26 shows the infrared spectrum of NPH30-SiO-25PEGio- In all situations shown in the figure the major silica bands are noticeable. This result evidences the presence of this main chemical entity that constitutes the fundamental structural unit of the nanoparticle. In the case of phenyl groups, their presence is also easily confirmed before and after functionalization and in the presence and absence of organic mold. Keeping the focus on polyethylene glycol, it is worth noting again the difficulty associated with observing the polymer bands overlapped by the silica bands.
  • event is 274 ° C referring to the decomposition of CTAB together with the polymeric entity cited.
  • the event mentioned shifts to 224 ° C, due to the thermal decomposition only of polyethylene glycol, which occurs in a smaller temperature range compared to the event involving the simultaneous presence.
  • PEG and CTAB This set of results is important to indicate the presence of the three organic species in NPH30-SiO-25PEGio, mainly by observing the displacement of decomposition events in the presence or absence of certain compounds.
  • thermogravimetric curves also presented in Figure 17 complement the above information regarding the chemical entities present in the molecular vehicle. Comparing the curve of NPH30-CTAB-SiOH with NPH30-CTAB-SiO-25PEG10, a new decomposition plateau is observed due to the PEG present. Comparing the curves of NPH30-SiOH and NPH30-SiO-25PEGio, both exempt from CTAB, it is evident that the thermal decomposition of polyethylene glycol between 217 ° C and 377 ° C is the only possible organic entity in this range. temperature, as CTAB is absent and, as already mentioned, the phenyl group decomposes from 324 ° C. In fact, it becomes complicated to estimate quantitatively the individual percentage of each chemical species commented, once in the temperature range between 324 ° C and 381 ° C CTAB, PEG and phenyl mass loss events occur simultaneously.
  • FIG. 18 shows the predominance of pores between 2 and 10 nm in both nanocarriers, classifying NPH30-SiO-25PEGio as a mesoporous material. Pore volumes indicated phenomenologically interesting values, that is, after functionalization the total pore volume ranges from 2.2 cm 3 / g to 1.74 cmVg. This result infers the possible spherical impedance of the pores by the superficially functionalized silanes, preventing the entry of gases inside the nanoparticle.
  • Figure 29 shows the 13 C NMR spectrum of the nanoparticle in which nuclei from the phenyl groups at 130.4 ppm (nucleus 1), 129 ppm (nucleus 2), 126.8 ppm (nucleus 3) are readily identified. ) and 134.1 ppm (core 4). It should be noted that said nuclear species are present before and after functionalization as expected. Regarding the identification of polyethylene glycol in the molecular carrier, one should identify the nucleus 7 peak of the polymer at 70.5 ppm in the NPH30-SiO-25PEGio spectrum.
  • NPH30-SiO-50PEGio The nanoparticle containing internal phenyl (30% mol-Si) groups as well as externally functionalized 50% mol-Si PEGió groups was named NPH30-SiO-50PEGio ⁇
  • Their respective structural characterizations showed satisfactory results according to pre-established expectations for the construction of said molecular vehicle.
  • Subtitles PC-SiOH, PC-50PEG10 and P-50PEG10 refer to NPH30-SiOH, NHPH30-CTAB-SiO-50PEG10 and NPH30-SiO-50PEG10. Initiating such studies by infrared vibrational spectroscopy whose spectrum is observed in Figure 21, the main silica bands were observed in all the synthetic steps presented.
  • Figure 22 shows the TG and DTA curves of NPH30-SiO-50PEG10.
  • Differential thermal analysis records an exothermic event at 267 ° C for NPH30-CTAB-SiO-50PEGio, indicating the thermal decomposition of surfactant and polyethylene glycol. After removal of the organic mold, the event is shifted to 221.7 ° C, associated only with the presence of the polymeric species in the absence of the soft template in this situation. From these considerations, observing the thermogravimetric curve of the NPH30-CTAB-SiOH allows visualization of CTAB mass loss starting at 135 ° C and ending at 381 ° C (22.2% mass).
  • the functionalization rates of the phenyl and PEG groups are consistent with the expected values.
  • the 28.7% mol Siphenyl graft is considerably close to the theoretical result, which is not the case with the polymeric entity whose 1.06% functionalization value, which is different from the theoretical value (50%), linked to possible effects. between the carbonic chains of the polymer which, in this system, interfere with the low reaction yield.
  • said functionalization content was sufficient to generate new chemical properties to the nanoparticle, such as improved colloidal stability and influence on hemolysis, topics discussed below.
  • the NPH30-SiO-50PEGio was subjected to nitrogen adsorption-desorption analysis whose isotherm is shown in Figure 23.
  • its nitrogen isotherm profile resembles that of the so-called nitrogen isotherm profile.
  • type IV with slight hysteresis of type H3 according to the IUPAC classification in the context of porous materials.
  • the observed pattern reflects a mesoporous material of complex porosity indicating a marked level of surface irregularities as the production of nahoparticles holding irregular surfaces.
  • the formation of a slight hysteresis at P / Po 0.40, typical of type IV isotherms for mesoporous materials, is notorious.
  • Nuclear Magnetic Resonance techniques are essential tools in the structural elucidation of organic and inorganic compounds. In this sense, it becomes necessary to complement the information obtained through infrared and thermal analysis techniques, as analyzed above. From the 13 C NMR of NPH30-SiO-50PEG10 Figure 24 shows the main peaks of the aromatic ring nuclei denominated 1 to 4 in chemical shifts of 130 ppm, 128.9 ppm, 127.4 ppm and 134.0 ppmError! Indicator not defined., Respectively, present at all stages as expected.
  • the nanoparticle functionalized with the GPS group showed barely noticeable changes in the profile of its spectrum. An enlargement of the bands close to 1250 cm -1 and 1070 cm -1 , typical of the. asymmetric stretches in the epoxy ring and asymmetric COC stretches, respectively. The changes are clearer compared to the spectrum of the NPH30-SiO-10PEGio, in which the bandwidth underwent slightly pronounced variations. After PEG coupling in the three-membered ring, an increase in bandwidth is observed in the region of 1100-1070 cm -1, where we observed the asymmetric COC stretching of polyethylene glycol. Although there is evidence of the functionalization of the glycidoxy group and the coupling of PEG in the molecular vehicle, it is necessary to use techniques such as thermal analysis and nuclear magnetic resonance to reach more robust conclusions.
  • Figure 33 shows the TG and DTA curves of NPH30-SiO-10GPS-PEG40 showing the complexity of events due to the presence of an intermediate component, the glycidoxy group, whose function is to condense on the external surface of the silica and act as a support for inserting polyethylene glycol in this region.
  • the starting nanoparticle TG and DTA curves present the release events of surfactants and phenyl groups in this order of occurrence, as shown in discussed in item 3.1.
  • the NPH30-CTAB-SIO-1 OGPS DTA peaks show a significant event at 300 ° C related to simultaneous release of CTAB and GPS.
  • the surface areas of NPH30-SiOH and NPH30-SiO-10GPS-PEG 4 o calculated by the BET method are 898 m 2 / g and 760 m 2 / g, respectively.
  • the total pore volume values calculated by the BJH method are respectively 2,2 cm 3 / g and 1,5 cm 3 / g.
  • the spatial predominance of cavities between 2 nm and 10 nm can be corroborated to the conclusion of the molecular vehicle complexity in the context of surface chemistry.
  • Figure 35 shows the carbon NMR of NPH30-SiO-10GPS-PEG40, as well as the intermediate and starting nanoparticles.
  • NPH30-SiOH spectrum the 4 peaks already mentioned are from the aromatic rings as the only organic entity present before functionalization. By condensing the glycidoxy group, changes in the spectrum become evident.
  • Figure 36 are shown the silicon NMR spectra on NPH30-SiOH and NPH30-SiO-10GPS. It should be noted that the silica structure before and after PEG40 coupling does not cover variations of areas between existing silicon sites, since the chemical reaction occurs between the polymer carboxylic acid and the epoxy group of the GPS function, ie is: silanol groups are not involved, eliminating the need for characterization of NPH30-SiO-IOGPS-PEG40 by 29 Si NMR, which would have the same profile as NPH30-SiO-10GPS. Analyzing the figure, silicon sites are typical in their respective chemical displacements, as commented earlier.
  • Table 1 shows the particle sizes and zeta potential of each molecular vehicle encompassed by the present invention.
  • Acceptable polydispersity indices (IPD) for a satisfactorily monodisperse system should be less than 0.4, which occurred in all situations. presented. It is observed that surface modification does not significantly alter the average size of colloids (as previously mentioned). Surface loads showed interesting differences from the point of view of surface modification. Knowing that the surface of NPH30-SiOH, as well as any silica nanostructures in basic medium has negative charges, it is expected that after PEG (neutral) functionalization there will be a tendency to surface neutrality even though such parameters are unreliable. to predict this surface phenomenon, since in this process there is a Gaussian distribution of loads and sizes.
  • the set of structural characterizations confirmed the achievement of all nanoparticles as well as the functionalizations on the internal (phenyl) and external (PEG) surfaces.
  • the materials showed large surface areas, important in the study of interaction of nanostructures with biosystems.
  • the instability of NPH30-SiOH is observed throughout the centrifuge's rotational range, since the percentage of this nanoparticle in the supernatant over the entire studied range was close to 0.
  • the Short-chair PEG functionalization content ranges from 10% mol-Si to 50% mol-Si.
  • An increased colloidal stability at 94 rcf is observed, ie, the NPH30-SiO-50PEGio has a higher percentage of nanoparticles suspended in relation to the others, confirming the direct relationship between the functionalization rate of PEGio silane and the stable colloidal characteristic.
  • NPH30-SiO-IOGPS-PEG40 becomes more stable compared to NPH30-SiO-10PEGio and more unstable compared to the others.
  • This result indicates that in the 10% functionalization content the nanoparticle holding longer chain PEG exhibits greater colloidal stability. From the rotation of 2348 rcf all nanoparticles exhibited unstable behavior as expected, since the centrifugal force acting in this situation is sufficient to generate coalescence. The influence of nanoparticle aggregation state as a function of time in hemolysis
  • the average size of nanoparticles in deionized water is approximately 100 nm according to the data commented on in the characterization topics. However, when added in PBS buffer the nanoparticles aggregate so that the average particle size reaches the order of 1000 nm (see graph below). This behavior is related to the strong electrostatic interactions between the negatively charged nanoparticles and the electrolytes present in the PBS buffer. In the time interval covered in this study, there was an increase in the average size of colloids as a function of time in all situations, reinforcing the gradual interaction between the colloidal system and the buffer solution in which it is dispersed. Note that the polydispersity index remained close to 1 in virtually all steps, reflecting significantly polydispersed systems.
  • the 10% mol-Si-containing PEGio silane nanoparticle has a larger surface area compared to the starting colloid and, according to 13 C NMR, the results also show that the amount of functionalized polyethylene glycol is significantly lower compared to those with 25% and 50% mol-Si, that is, the NPH30-SiOH and NPH30-SiO-10PEGio have significant structural similarities, differing only by the tiny presence of PEGio for the latter.
  • this molecular carrier has the second smallest surface area in relation to the nanoparticles of the summed to the high PEG content grafted to its surface.
  • the smaller surface area as well as the aggregating effect are factors that directly contribute to the minimization of hemolysis in the context of mesoporous silica nanoparticles.
  • the graph reflects such assumptions by observing the optimal performance of the NPH30-SiO-50PEGio with only 1.6% and 1.18% hemolysis at times 1 minute and 60 minutes, respectively.
  • the epoxy ring of said functions can be opened to form a diol group which, like silanol functions, would interact with red blood cells in a similar manner causing toxicity and thus acquiring substantially the same behavior as NPH30-SiOH.
  • this molecular vehicle exhibited behavior that directs the results to the reduction of hemolysis, indirectly indicating the presence of PEG40 in its structure. If this polymer is present then it should necessarily be covalently attached to the nanoparticle and therefore the peak in its 13 C NMR spectrum is from carbon 18.
  • the ethanol curcumin calibration curve was developed at five known concentrations (1, 5, 10, 30 and 50 ⁇ g / mL). For each concentration, the area under the curcumin absorption curve in the region between 200 and 550 nm was estimated for graph acquisition relating the area as a function of concentration. Finally, to determine the concentration of encapsulated curcumin in each of the pegylated silica nanoparticles in the above mentioned situations, 1 ml of the respective supernatant was subjected to UV-Vis spectroscopy so that area value under the curve similarly estimated As described in the paragraph above, it was compared to the calibration curve and concentration-related, thus acquiring the drug mass value per unit volume of suspension.
  • the equation of the line is described as:
  • Curcumin concentration (x) [area under the curve (y) - 22.9] / 14.3
  • Figure 41 shows the graph of curcumin concentration in the pegylated silica nanoparticles after each separation by centrifugation and decantation.
  • the influence of PEG on curcumin encapsulation efficiency is noticeable, since all pegylated nanoparticles comprise a higher concentration of encapsulated drug over non-functionalized nanoparticle (NPH30-SiOH).
  • NPH30-SiOH non-functionalized nanoparticle
  • curcumin when added to the described silica nanoparticle suspensions, curcumin is not only encapsulated in nanocarriers, but also possibly interacting with the outer surface of molecular vehicles. That is, the drug referred to as encapsulated in any context described above may also be interacting superficially through specific interactions to be confirmed in further work and in greater detail.
  • Figure 42 shows the image of the suspensions NPH30 SiOH-silica nanoparticles NPH30-10PEG 5 oo o NPH30-10PEG NPH30-50PEG 50 'in 5ooo. presence of curcumin and said drug added in water in the absence of molecular vehicles (H2O) after 24 hours of decantation and prior to removal of the supernatant.
  • H2O molecular vehicles
  • the hydrophobic characteristic of the drug is evident in the clear supernatant in the H2O bottle characterizing the problem of the insolubility of hydrophobic drugs in the blood (hydrophilic) whereas in the presence of silica nanoparticles their yellow colored supernatants show the stability of the suspended drug upon application of the drugs. molecular vehicles.

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Abstract

La présente invention concerne un procédé d'obtention de nanoparticules de silice pégylées transporteuses de substances médicamenteuses hydrophobes, les nanoparticules de silice obtenues ou non au moyen dudit procédé et leur utilisation dans le traitement du cancer. La substance médicamenteuse peut être maintenue efficacement à l'intérieur desdites particules. On évite ainsi sa libération précoce dans le milieu extérieur pendant le transport intraveineux, d'où une réduction des effets secondaires associés à la chimiothérapie et une réduction de la concentration posologique nécessaire pour le traitement antitumoral.
PCT/BR2015/000202 2014-12-16 2015-12-16 Procédé d'obtention de nanoparticules de silice pégylées transporteuses d'agences pharmaceutiques hydrophobes, nanoparticules ainsi obtenues et leurs utilisations WO2016094991A1 (fr)

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BRBR1020140316884 2014-12-16
BR132015030941A BR132015030941F1 (pt) 2015-12-10 2015-12-10 Processo de obtenção de nanopartículas de sílica peguiladas carreadoras de fármacos hidrofóbicos, nanopartículas assim obtidas e seus usos
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US11191745B2 (en) 2017-02-03 2021-12-07 City Of Hope Silica nanoparticle with an insoluble drug

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