Method for obtaining biodegradable colloidal particles
By preparing colloidal particles that combine biodegradable anionic and cationic polysaccharides, the high cost and drug leakage problems of existing nanoscale drug delivery systems have been solved, achieving biocompatibility and stability, and improving drug bioavailability and targeting.
Patent Information
- Application Number
- CN202380062455.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2022-06-29
- Filing Date
- 2023-06-28
- Publication Date
- 2025-12-12
AI Technical Summary
Existing nanoscale drug delivery systems using nanoparticles prepared from synthetic polymers suffer from high costs, difficulties in industrial production, and drug leakage issues, and require the avoidance of toxic solvents and surfactants.
Colloidal particles were prepared by combining biodegradable anionic and cationic polysaccharides. The particles were formed with an average size of less than 500 nm by electrostatic attraction. The preparation process avoided the use of surfactants and chemical crosslinking agents, and the particle size was controlled by ultrasonic treatment and centrifugation techniques.
A highly biocompatible and easily manufactured nanoscale drug delivery system has been developed, which can be stably stored and transported, rapidly biodegraded, and improves drug bioavailability and targeting.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
TECHNICAL FIELD
[0001] The present invention relates to inert carriers or additives physically combined with active ingredients, and more particularly, it relates to biodegradable colloidal particles and a process for their extraction. BACKGROUND
[0002] Drug delivery systems are now as important as the drugs themselves. Controlled release provides sustained dosing of a drug while maintaining its blood concentration within the therapeutic limit. Thus, a drug delivery system can influence the pharmacological activity by modulating its release from the carrier. Other advantages include improved patient compliance (reduced dosing frequency), non-invasive route of administration, minimization of local and systemic side effects, and thus reduced toxicity profile. Nanoscale drug delivery systems can deliver drugs to the site of action in a pre-designed manner, thus minimizing side effects and improving the bioavailability of the drug. For example, colloidal drug carriers such as micelles, liposomes, nanoparticles and emulsions are used to increase the concentration of drugs that enter the brain through the blood-brain barrier. Furthermore, active ingredients entrapped in drug carrier systems can be protected from enzymatic degradation.
[0003] The ability of these systems to cross external barriers and enter the interior of the organism depends on their size and composition. Nanoscale particles increase the degree of transport compared to larger particles. Furthermore, if they are prepared from naturally occurring and biocompatible polymers, their chances of being naturally transported through the mucosae of the body by known transport mechanisms without altering the physiology of the epithelium increase. However, the main limitations of nanoparticles prepared from synthetic polymers are the high costs incurred and the production techniques that are not convenient for industrial scale production, the toxic solvents used in the production process, and the leakage of the drug before reaching the target area. To overcome these limitations, research has focused on finding new methods to obtain novel biodegradable nanoparticles from naturally occurring and biocompatible polymers.
[0004] For example, Katuwavila et al. in their publication "Chitosan-Alginate Nanoarticle System Efficiently Delivers Doxorubicin to MCF-7 Cells, Journal of Nanomaterials" describe a method for obtaining alginate-chitosan-doxorubicin nanoparticles by dropwise addition of a mixture of chitosan (2 mg / mL, pH 4.8) and the surfactant Tween 80 to a solution of alginate (1 mg / mL, pH 5.2) previously mixed with doxorubicin (DOX). Although both chitosan and alginate are reported as pharmaceutically acceptable natural polymers for targeted drug delivery, their use alone or in combination involves the use of surfactants, chemical cross-linking agents and / or organic solvents, which can alter the properties of the active ingredient to be administered, even increasing the production costs.
[0005] Therefore, there is a need to find new methods to obtain active ingredient release systems that allow efficient integration into the biological system and that the particles are rapidly biodegradable in the biological environment. Moreover, these systems should be easy to produce and stable during storage and transport. SUMMARY
[0006] To this end, a first aspect of the present invention relates to a system for the release of a biologically active compound comprising biodegradable colloidal particles, in turn comprising an anionic polysaccharide, a cationic polysaccharide and at least one biologically active compound, having an average size of less than 500 nm.
[0007] A second aspect of the present invention relates to a method for obtaining biodegradable colloidal particles, comprising the steps of: (a) estimating the stoichiometric charge ratio between the anionic polysaccharide and the cationic polysaccharide according to the degree of deesterification of the anionic polysaccharide and the degree of deacetylation of the cationic polysaccharide; (b) preparing a solution of the anionic polysaccharide and adjusting to an acidic pH according to the stoichiometric charge ratio; (c) preparing a solution containing the cationic polysaccharide and adjusting to an acidic pH according to the stoichiometric charge ratio; (d) adding the solution containing the cationic polysaccharide to the solution containing the anionic polysaccharide to obtain biodegradable colloidal particles; (e) reducing the particle size of the biodegradable colloidal particles; (f) separating the biodegradable colloidal particles from the supernatant; (g) resuspending the biodegradable colloidal particles and storing them. BRIEF DESCRIPTION OF DRAWINGS
[0008] The novel aspects considered characteristic of the invention will be particularly pointed out in the annexed claims. However, the characteristics and advantages will be better understood in the following examples, when read in conjunction with the drawings, in which:
[0009] Figure 1 FTIR-ATR spectrophotogram of chitosan is shown, with a resolution of 0.5 cm -1 . The peaks at 1320 cm -1 and 1420 cm -1 are indicated with arrows and they correspond to N-acetyl and N-amino groups, respectively.
[0010] Figure 2 Results of the hydrodynamic diameter (D H , intensity average) in nanometers of biodegradable chitosan / alginate colloidal particles prepared with different charge ratios are shown.
[0011] Figure 3 Zeta potential obtained for biodegradable chitosan / alginate colloidal particles prepared with different charge ratios is shown.
[0012] Figure 4 Biodegradable chitosan / alginate colloidal particles prepared with different charge ratios analyzed by Atomic Force Microscopy (AFM) are shown.
[0013] Figure 5 Results of the hydrodynamic diameter (D H , intensity average) in nanometers obtained for biodegradable chitosan / alginate colloidal particles subjected to different pH values are shown.
[0014] Figure 6 Results of the polydispersity index (PDI) obtained for biodegradable chitosan / alginate colloidal particles subjected to different pH values are shown.
[0015] Figure 7 Results of the Zeta potential obtained for biodegradable chitosan / alginate colloidal particles subjected to different pH values are shown.
[0016] Figure 8 Hydrodynamic diameter (D H , intensity average) in nanometers of biodegradable chitosan / alginate colloidal particles obtained using different surfactants and experimental conditions is shown.
[0017] Figure 9 Mass of DOX-HCl (Doxorubicin hydrochloride) encapsulated in biodegradable chitosan / alginate colloidal particles of different charge ratios is shown.
[0018] Figure 10 Encapsulation efficiency (EE%) of DOX-HCl in biodegradable chitosan / alginate colloidal particles of different charge ratios is shown.
[0019] Figure 11The mass of ICG encapsulated in biodegradable chitosan / alginate colloidal particles with different charge ratios is shown.
[0020] Figure 12 The encapsulation efficiency (EE%) of ICG in biodegradable chitosan / alginate colloidal particles with different charge ratios is shown.
[0021] Figure 13 Heating curves of ICG-functionalized chitosan / alginate biodegradable colloidal particles are shown, where DT represents the recorded temperature difference. The laser power was 2 W. The line corresponds to the fit of the Roper model, and the points correspond to the experimental data.
[0022] Figure 14 The results of atomic force microscopy (AMF) of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Test 1 are shown in the scanning regions of 2500 nm x 2500 nm (left) and 5000 nm x 5000 nm (right). The particles have an intensity-average hydrodynamic diameter of 339.0 nm and a zeta potential of -29.4 mV.
[0023] Figure 15 The results of atomic force microscopy (AMF) of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Test 2 are shown in the scanning regions of 2500 nm x 2500 nm (left) and 5000 nm x 5000 nm (right). The particles have an intensity-average hydrodynamic diameter of 358.5 nm and a zeta potential of -41.4 mV.
[0024] Figure 16 The results of atomic force microscopy (AMF) of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Test 3 are shown. The particles have an intensity-average hydrodynamic diameter of 346.0 nm and a zeta potential of -39.5 mV.
[0025] Figure 17 The calibration curve of DOX-HCl obtained from the UV-Vis spectrophotometer is shown, from which the equation is obtained: y = 28.588x + 0.0145, where R0 2 = 0.99.
[0026] Figure 18 This study compares the encapsulation efficiency (EE%) obtained by DOX-HCl functionalization in biodegradable chitosan oligosaccharide / alginate colloidal particles using two different methods.
[0027] Figure 19The ICG calibration curve obtained from the UV-Vis spectrophotometer is shown, from which the equation is obtained: y = 39.704x + 0.105, R 2 = 0.99.
[0028] Figure 20 This study compares the encapsulation efficiency (EE%) and drug loading percentage obtained by ICG functionalization in biodegradable chitosan oligosaccharide / alginate colloidal particles using two different methods. Detailed Implementation
[0029] This invention presents several advantages over existing technologies, among which we can mention the system for releasing bioactive compounds, comprising biodegradable colloidal particles that allow for safe integration into biological systems and rapid biodegradation due to the natural properties of the polysaccharides used in its preparation. Furthermore, its extraction method eliminates the need for surfactants, chemical cross-linking agents, and / or organic solvents, which facilitates their production process and ensures their stability during storage and transportation.
[0030] Therefore, the present invention relates first to a system for releasing a bioactive compound comprising biodegradable colloidal particles, which in turn comprise anionic polysaccharides, cationic polysaccharides and at least one bioactive compound having an average size of less than 500 nm.
[0031] Preferably, the anionic polysaccharide is alginate. The cationic polysaccharide is selected from chitosan, chitosan oligosaccharide, or a mixture thereof.
[0032] More preferably, the cationic polysaccharide is chitosan oligosaccharide.
[0033] Anionic polysaccharides and cationic polysaccharides bind together through electrostatic attraction.
[0034] Preferably, the bioactive compound has antitumor activity and is photosensitizing.
[0035] A second aspect of the present invention relates to a method for obtaining biodegradable colloidal particles, the method comprising the following steps: (a) estimating the stoichiometric charge ratio between anionic and cationic polysaccharides based on the degree of deesterification of the anionic polysaccharide and the degree of deacetylation of the cationic polysaccharide; (b) preparing a solution containing anionic polysaccharides based on the stoichiometric charge ratio and adjusting it to an acidic pH; (c) preparing a solution containing cationic polysaccharides based on the stoichiometric charge ratio and adjusting it to an acidic pH; (d) adding the solution containing cationic polysaccharides to the solution containing anionic polysaccharides to obtain biodegradable colloidal particles; (e) reducing the particle size of the biodegradable colloidal particles; (f) separating the biodegradable colloidal particles from the supernatant; and (g) resuspending and storing the biodegradable colloidal particles.
[0036] Preferably, the anionic polysaccharide is alginate.
[0037] Preferably, the cationic polysaccharide is selected from chitosan, chitosan oligosaccharide, or a mixture thereof. More preferably, the cationic polysaccharide is chitosan oligosaccharide.
[0038] Preferably, the solvent used to prepare the polysaccharide solution is water.
[0039] Preferably, the degree of deesterification in step (a) is determined by potentiometric titration or conductivity titration.
[0040] Preferably, the degree of deacetylation in step (a) is determined by nuclear magnetic resonance (NMR), ultraviolet-visible spectrophotometry, potentiometric titration or conductivity titration, or Fourier transform attenuated total reflectance infrared spectroscopy (FTIR-ATR) spectrophotometry.
[0041] Preferably, the pH of the anionic polysaccharide solution is adjusted to 4.
[0042] Preferably, the pH of the cationic polysaccharide solution is adjusted to 5.
[0043] Preferably, the cationic polysaccharide solution is filtered before being used in step (d).
[0044] Preferably, step (d) is carried out by continuous dripping with vigorous stirring.
[0045] Preferably, the particle size reduction in step (e) is achieved by ultrasonic treatment of the biodegradable colloidal particles: the particles are placed in a container of ice water to relieve overheating for 5-10 minutes.
[0046] Preferably, the separation in step (f) is performed by centrifugation at a speed of 6000-7000 rpm.
[0047] Preferably, the resuspension in step (g) is performed by vortexing for at least 10 minutes.
[0048] Preferably, the method further includes the additional step of functionalizing the biodegradable colloidal particles with at least one bioactive compound.
[0049] Preferably, the functionalization step is performed before step (d), wherein the solution of the cationic polysaccharide obtained in step (e) is mixed with a solution having at least one bioactive compound.
[0050] Preferably, the functionalization step is performed after step (g) and further comprises the following steps: (i) adding a solution having at least one bioactive compound to a container containing biodegradable colloidal particles; (ii) adding deionized water to the mixture; (iii) stirring; (iv) centrifuging; (v) resuspending the previously functionalized biodegradable colloidal particles in deionized water to remove uncaptured bioactive compounds; and (vi) freeze-drying the functionalized biodegradable colloidal particles.
[0051] More preferably, the stirring in step (iii) is carried out on a vortex mixer for at least 1 hour.
[0052] More preferably, the centrifugation in step (iv) is carried out at a temperature of at least 10 °C and at 6000-7000 rpm for 15-30 minutes.
[0053] More preferably, the bioactive compound has antitumor activity and is photosensitizing.
[0054] The term "photosensitizer" refers to a bioactive compound used in photodynamic therapy.
[0055] It refers to the fact that when it is present in the body, it can trigger a skin reaction through interaction with ultraviolet radiation, that is, it is sensitive to sunlight.
[0056] The advantages of the present invention will be better understood from the following embodiments, which are given for illustrative purposes only to allow for a full understanding of the preferred embodiments of the invention and do not imply that there are no other embodiments, not shown, that can be practiced based on the above detailed description. Example
[0057] Example 1
[0058] Experiments were conducted according to the principles of the invention to illustrate a method for obtaining biodegradable colloidal particles from polysaccharides, particularly chitosan and alginate.
[0059] First, to determine the charge ratio used for particle preparation, the deesterification rate of alginate was assumed to be 100%, a figure that was subsequently revised. Using this assumption, the negative charge density of alginate (Cat: A1112) is d. - = 5.16 x 10 -3The molar negative charge per gram of alginate was determined. The degree of deacetylation (DD%) of low molecular weight chitosan (Mw ~ 125000 g / mol) was then measured to determine the charge ratio for preparing biodegradable colloidal particles. DD% represents the molar percentage of glucosamine monomer units relative to the total monomer groups (i.e., glucosamine units plus N-acetylglucosamine units). Therefore, DD% is translated as the concentration of -NH2 groups per unit mass of chitosan. Various techniques exist for determining DD%, including NMR, UV-Vis spectrophotometry, potentiometric titration, conductivity titration, and FTIR-ATR spectrophotometry. In this embodiment, FTIR-ATR spectrophotometry was employed due to its speed, accuracy, and simplicity. Approximately 500 mg of chitosan powder (Cat: 448869-250G) was placed in an attenuated total reflectance (ATR) optical reader with a resolution of 0.5 cm⁻¹. -1 The spectrum was captured and repeated 30 times to improve peak clarity. The results are as follows: Figure 1 As shown. Subsequently, baseline subtraction of the spectrum was performed using Origin 8.0® software, and the result was obtained at 1320 cm⁻¹. -1 and 1420 cm -1 The absorbance was measured at the ratio of N-acetyl groups to N-amino groups, respectively.
[0060] DD% is obtained from the degree of acetylation (DA%) using the following formula:
[0061]
[0062] For chitosan, the absorbances obtained at 1320 nm and 1420 nm were 0.02689 and 0.02343, respectively. Therefore, DA% = 24.43% and DD% = 75.57%, which are within the range reported by the supplier (DA% ≥ 75%). Based on the average molecular weight Pm = 120000 g / mol provided by the supplier, the positive charge density in chitosan was estimated to be d. + = 3.97 x 10 -3 Molar positive charge / gram chitosan.
[0063] The relationship (R) between the negative and positive charges of each alginate / chitosan mixture was determined using the following formula:
[0064]
[0065] Among them, V A C is the volume of the alginate solution. A d represents the mass concentration of the alginate solution. -This represents the number of negative charges per gram of alginate. The same definition applies to the variables in the denominator of the relation, but specifically to chitosan.
[0066] Once the charge ratio was determined, a polysaccharide solution was prepared and the pH was adjusted. Sodium alginate was adjusted to pH 4, and chitosan to pH 5, before mixing. The solution was filtered using a 0.45-micron filter membrane to remove impurities.
[0067] To prepare the colloidal particles, a mixing method was employed, involving dropwise addition from one solution to another with constant and vigorous stirring. After mixing the biopolymer, the mixture was sonicated for 5–10 minutes using a Fisherbrand™ 120 Sonic Dismembrator. The container containing the mixture (50 mL conical tube or 20 mL glass bottle) was placed in an ice-water bath to prevent overheating of the sample and potential damage to the bioactive compound to be encapsulated. Finally, the sample was washed by centrifugation. The resulting colloidal particles were centrifuged at 6000 rpm, the supernatant was recovered, and the precipitate was resuspended by vortexing.
[0068] Figure 2 The hydrodynamic diameter (D) is shown in nanometers. H The result of the average intensity, and Figure 3 The zeta potentials obtained for formulations with different charge ratios are shown (these charge ratios are estimated because they assume 100% deesterification of alginate and use the average molecular weight provided by the supplier). In the developed method, the alginate solution was set as the "receiver," i.e., it was kept under constant stirring while chitosan oligosaccharide was added dropwise onto it. It can be seen that the hydrodynamic diameter decreases with increasing charge ratio (R), achieving a hydrodynamic diameter as high as 184.4 ± 1.3 nm when the ratio R = 30. Figure 3 As shown, the Zeta potential changes from positive to negative when the ratio approaches 1. That is, the effective isoelectric point of the colloidal particles is near this ratio. The above results are consistent with the particle size data, because when the value approaches R=1, the size increases due to decreased electrostatic stability and particle aggregation.
[0069] Some samples were analyzed using atomic force microscopy (AFM) to obtain diameter distribution, such as Figure 4 As shown. For example, for a sample with R = 4.2, an average diameter of 135 ± 18 nm was obtained. This average value was obtained by analyzing AFM images using ImageJ® software. In colloidal particles obtained from polysaccharides with high water absorption capacity, Figure 4 The diameter obtained is smaller than the hydrodynamic diameter obtained using the dynamic light scattering technique (Zetasizer).
[0070] To evaluate the colloidal stability of the particles (R=3) at different pH values, samples were prepared by mixing 1 mL of particles with 1 mL of 50 mM NaCl, and the pH was adjusted to different values using HCl or NaOH. Hydrodynamic diameter, polydispersity index (PDI), and zeta potential were measured at different pH values. Figure 5 , 6 Figures 7 and 8 show the results obtained. The results indicate that the hydrodynamic diameter remains stable within the pH range of 4–7, even exhibiting a slight decrease in size at pH = 8, attributed to the remodeling of the alginate and chitosan polymer chains. At the more alkaline pH values of 10 and 12, a slight increase in hydrodynamic diameter was observed, but not to the point of drastic instability of the colloidal suspension. Conversely, at pH = 2, instability of the colloidal particles was observed, manifested as a significant increase in diameter to values greater than 2700 nm. This instability is due to the protonation of the alginate carboxyl groups, i.e., a decrease in the Z-potential, leading to particle aggregation and sedimentation.
[0071] This behavior allows us to infer that the configuration or structure of particles with this composition is likely an internal region or core rich in chitosan chains, surrounded by a crown with a high concentration of alginate. Because the particles have a pH greater than 3 (the pKa of sodium alginate is 3.3–3.6), they are stabilized by electrostatic repulsion through their electric double layer. When the pH decreases below the pKa of alginate, the carboxyl groups are protonated, and the colloidal particles lose their negative charge, as... Figure 7 As shown, the Zeta potential value changed from below -30 mV to -5 mV, which is very close to neutral, triggering particle aggregation.
[0072] In developing a method for preparing colloidal particles, different strategies were tested. The goal was to obtain particles smaller than 1000 nm. Initially, attempts were made to prepare them by dropwise addition of an alginate solution to a chitosan solution without sonication or adjustment of the pH of the precursor solution. This resulted in gels larger than 1 mm, visible to the naked eye as precipitates. At this point, a dropwise addition method using an infusion pump, combined with sonication, was adopted. The result was a reduction in size, but still larger than 1000 nm. Reviewing existing literature, it was found that some authors used stabilizers to control the size of micron- and nano-sized gels. This strategy was tried, yielding particles with a hydrodynamic diameter of approximately 800 nm, such as... Figure 8As shown. The smallest particles were prepared by adjusting the pH, sonicating for 10 minutes, and using a solution containing Pluronic F127 (stabilizer). Subsequently, it was decided to filter the chitosan oligosaccharide solution before mixing it with alginate by dropwise addition. The purpose of this filtration was to remove large visible aggregates and other impurities. The solution was filtered through a 0.45-micron filter, resulting in a clear, aggregate-free, and slightly viscous solution. It is also possible that large polymer chains of chitosan may be removed during this filtration. Therefore, larger chains are likely to be removed when filtering the chitosan solution, which is reflected in the production of particles with an average diameter of less than 500 nm.
[0073] Example 2
[0074] An assay was performed in which biodegradable colloidal particles of the natural biopolymer obtained in Example 1 were functionalized with doxorubicin hydrochloride (DOX-HCl) and indocyanine green (ICG), respectively, to measure their function as delivery and release systems for bioactive compounds.
[0075] Due to their physicochemical properties, particularly their high density of positive and negative charges and their gel-like structure, biodegradable colloidal particles of alginate and chitosan possess a strong ability to encapsulate and / or capture positively and negatively charged bioactive compounds. To evaluate the ability to capture antitumor drugs and photosensitizing drugs, the particles were incubated with doxorubicin hydrochloride (DOX-HCl) and indocyanine green (ICG) solutions, respectively, and the amount of drug captured was quantified by comparing a control sample with deionized water without the particles.
[0076] Colloidal particles of alginate and chitosan with a charge ratio > 2 and a zeta potential < -20 mV were incubated at room temperature for 24 hours with moderate shaking to capture large amounts of DOX-HCl. Figure 9 and Figure 10 The amount of DOX-HCl and encapsulation efficiency (EE%) of particles with different charge ratios are shown.
[0077] Similarly, preliminary ICG charge tests were performed on a particle sample with R = 7 and a Zeta potential of -39.4 mV, and another sample with R = 1.5 and a Zeta potential of +30.1 mV. Samples were prepared by adding 50 μL of 12.9 mM ICG solution to 1 mL of colloidal particles. A control sample was prepared using an equal volume of ICG, i.e., 50 μL of 12.9 mM ICG solution added to 1 mL of deionized water. Two samples with similar Zeta potentials but different charges were selected; one sample had a negative surface charge, and the other had a positive surface charge. Figure 11 and Figure 12The ICG content and encapsulation efficiency (EE%) of particles with different charge ratios are shown. It was observed that the sample with R = 1.5 and Zeta potential = +30.1 mV captured 1.8 times more ICG than the negatively charged sample. This is due to the negative charge of the ICG from its ionized sulfonic acid groups and its electrostatic interaction with the positive charge of the colloidal particles. Colloidal particles containing ICG can be used as photodynamic and photothermal therapy (phototherapy) systems. Preliminary results of the particle photothermal performance determination are presented below, specifically determining their photothermal efficiency by fitting the temperature rise data as a function of the 800 nm wavelength laser irradiation time. The data are consistent with the model reported by Roper et al. (Transfer Transduced by Surface Plasmon Resonant Gold Nanoparticles, The Journal of Physical Chemistry C 111(9) (2007) 3636-3641); and follow the method reported by Almada et al. (Photothermal conversion efficiency and cytotoxic effect of gold nanorods stabilized with chitosan, alginate and poly(vinyl alcohol), Materials Science and Engineering: C 77 (2017) 583-593).
[0078] Photothermal efficiency was measured as follows: 2 mL of ICG particles were added to a quartz cell, and the solution was stirred to mix the sample and induce a uniform temperature rise. The sample was irradiated with an 800 nm laser at different powers (1 W, 1.5 W, and 2 W) for 15 minutes, followed by 5 minutes without irradiation. The heating curve using 2 W irradiation is shown below. Figure 13 As shown in the diagram. Temperature is measured via a type K thermocouple connected to a digital thermometer, and recorded every 30 seconds. The quartz cell is insulated to prevent solution evaporation and heat loss.
[0079] The same treatment was applied to water to obtain the parameter "Q0", which was calculated by irradiating water with a resistivity of 18.2 MΩ at the same power as the biodegradable colloidal particles for 15 minutes. Furthermore, ICG-free biodegradable colloidal particles were measured to compare their photothermal effects. The characteristic rate constant t was analyzed. s The maximum temperature rise was observed to occur within the first 4-5 minutes of irradiation, after which the rate of increase decreased, such as... Figure 13As shown. The presence of ICG in biodegradable colloidal particles endows them with photoresponsive properties, which depend on the intensity of the light stimulus and the concentration of the compound in the particles. ICG-containing particles exhibit a significant temperature increase, with a maximum thermal efficiency of 50.68% and a obtained ΔT... max The temperature was 13.3 degrees Celsius. Thermal efficiency was shown to be strongly dependent on irradiation power; furthermore, the temperature rise could be controlled by varying the combination of the number of particles per unit volume and the laser power.
[0080] Example 3
[0081] An assay was performed to demonstrate a method for obtaining biodegradable colloidal particles from polysaccharides (particularly chitosan oligosaccharides and alginate) according to the principles of the present invention.
[0082] Based on results obtained with low molecular weight chitosan (Mw ~ 125000 g / mol) (its solution passed through a 0.45 μm filter), similar results were attempted using chitosan oligosaccharide (Mw ~ 5000 g / mol), aiming to achieve the same effect without the need for filtration through such a small pore size, thus reducing workload and cost. Particles were prepared using three different volume ratios of chitosan oligosaccharide and alginate solutions: 2.5 mL alginate and 5 mL chitosan oligosaccharide (Experiment 1), 5 mL chitosan oligosaccharide and 2.5 mL alginate (Experiment 2), and 5 mL alginate and 5 mL chitosan oligosaccharide (Experiment 3). After the chitosan oligosaccharide solution was added dropwise to the alginate solution, all samples were sonicated at 80% amplitude for 5 minutes in an ice bath. For atomic force microscopy (AFM) imaging, samples were centrifuged at 9000 rpm for 30 minutes at 10 °C. The supernatant was discarded, and the samples were resuspended by pipetting and then sonicated in the bath for 2 minutes.
[0083] Figure 14 Atomic force microscopy (AMF) results of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Experiment 1 are shown in scanning regions of 2500 nm x 2500 nm (left) and 5000 nm x 5000 nm (right), where the particles exhibit an intensity-averaged hydrodynamic diameter of 339.0 nm and a Zeta potential of -29.4 mV. On the other hand, Figure 15 Atomic force microscopy (AMF) results of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Experiment 2 are shown in scanning regions of 2500 nm x 2500 nm (left) and 5000 nm x 5000 nm (right), where the particles exhibit an intensity-averaged hydrodynamic diameter of 358.5 nm and a Zeta potential of -41.4 mV. Finally, Figure 16Atomic force microscopy (AMF) results of the biodegradable chitosan oligosaccharide / alginate colloidal particles obtained in Experiment 3 are shown in the scanning regions of 2500 nm x 2500 nm (left) and 5000 nm x 5000 nm (right), where the particles exhibit an intensity-averaged hydrodynamic diameter of 346.0 nm and a Zeta potential of -39.5 mV.
[0084] Example 4
[0085] Two different methods were used in the experiments in which biodegradable colloidal particles derived from natural alginate / chitosan oligosaccharide (chitosan molecular weight = 5000 g / mol) were functionalized with doxorubicin hydrochloride (DOX-HCl): (i) functionalization of previously prepared biodegradable colloidal particles with DOX-HCl; and (ii) functionalization of biodegradable colloidal particles during gelation. The aim was to evaluate their function as delivery and release systems for bioactive compounds.
[0086] In the first method (i), particles were prepared using a volume ratio of 5 mL alginate (1.5 mg / mL) to 5 mL chitosan oligosaccharide (1 mg / mL). The preparation and rinsing methods were as previously described. After rinsing, the particles were processed as follows: (i) 0.5 mL of particles were placed in a 1.5 mL conical tube; (ii) 10 µL, 20 µL, 30 µL, 40 µL, or 50 µL of DOX-HCl solution (1 mg / mL) was added; (iii) the necessary amount of deionized (DI) water was added to each tube to bring the total volume to 1 mL; (iv) they were stirred in a vortex mixer for 1 hour; (v) they were centrifuged at 9000 rpm for 30 minutes at 10 °C; (vi) the supernatant was removed and the particles were purified by... Figure 17 The amount of uncaptured DOX-HCl was determined using a calibration curve obtained with a UV-Vis spectrophotometer, where the equation was: y = 28.588x + 0.0145, with R² = 0.99; (v) the mass of DOX-HCl captured, encapsulation efficiency (EE%), and percentage of DOX-HCl encapsulated in each sample were determined. Table 1 shows the results of DOX-HCl encapsulation in alginate / chitosan oligosaccharide particles.
[0087]
[0088] In the second method (ii), during gelation, a DOX-HCl solution was mixed with an alginate solution, and then chitosan oligosaccharide was added as previously described. The procedure was as follows: (i) 100 µL of DOX-HCl solution (1 mg / mL) was added to 5 mL of alginate (1.5 mg / mL); (ii) a chitosan oligosaccharide solution (1 mg / mL) was added dropwise to the alginate solution; (iv) the particles were centrifuged at 9000 rpm for 30 minutes at 10 °C; (v) the concentration of DOX-HCl in the supernatant was determined to ascertain the mass of uncaptured DOX-HCl; and (vi) the mass, EE%, and DOX-HCl% of the captured DOX-HCl were calculated. Table 2 shows the results obtained from encapsulating DOX-HCl in the alginate / chitosan oligosaccharide particles.
[0089]
[0090] Figure 18 This paper compares the encapsulation efficiency (EE%) obtained by DOX-HCl functionalization of biodegradable chitosan oligosaccharide / alginate colloidal particles using two methods. It is important to emphasize that although a higher encapsulation efficiency percentage and a larger mass of captured DOX-HCl were achieved in the first method (i), the release kinetics in the samples prepared by the two methods are likely to be very different. Since DOX-HCl may adsorb on the surface, a much faster release is expected in the sample prepared by the first method (i), while a slower release is likely in the sample prepared by the second method (ii), where DOX-HCl is expected to reside in more of the internal region of the particle.
[0091] Example 5
[0092] Two different methods were used in the experiment to functionalize biodegradable colloidal particles derived from natural alginate / chitosan oligosaccharide polysaccharide (chitosan molecular weight = 5000 g / mol) with indocyanine green (ICG): (i) ICG functionalization using previously prepared biodegradable colloidal particles; and (ii) ICG functionalization during the gelation process of the biodegradable colloidal particles. The aim was to evaluate their function as an application and release system for bioactive compounds. In the first method, in which ICG was adsorbed onto pre-prepared particles, biodegradable colloidal particles were prepared at a volume ratio of 2.5 mL alginate (1.5 mg / mL) to 5 mL chitosan oligosaccharide (1 mg / mL). The preparation and washing methods were performed according to the previously described procedures. Once the particles have been rinsed, proceed as follows: (i) Place 0.5 mL of the biodegradable colloidal particles into a 1.5 mL conical tube; (ii) Add 10 µL, 20 µL, 30 µL, 40 µL, or 50 µL of ICG solution (5 mg / mL) to the 1.5 mL conical tube; (iii) Add the necessary amount of deionized (DI) water to each tube to bring the total volume to 2 mL; (iv) Vortex mix them for 1 hour; (v) Centrifuge them at 15,000 rpm for 15 minutes at 10 °C; (vi) Remove the supernatant and pass it through a vortex mixer as follows: Figure 19 The calibration curve shown is used to determine the amount of uncaptured ICG using a UV-Vis spectrophotometer, where the equation obtained is: y = 39.704x + 0.105, where RV 2 = 0.99; (vii) Calculate the mass of captured ICG, encapsulation efficiency (EE%), and percentage of ICG encapsulation (ICG%) for each sample. Table 3 shows the results of ICG encapsulation in alginate / chitosan oligosaccharide particles.
[0093]
[0094] Unlike the methods described above, in the second encapsulation method, during gelation, the ICG solution is mixed with the chitosan oligosaccharide solution, and then the mixture is added to the alginate as previously described. The procedure was as follows: (i) 200 µL of ICG solution (5 mg / mL) was added to 5 mL of chitosan oligosaccharide (1 mg / mL); (ii) the mixture was added dropwise to a solution containing 2.5 mL of alginate (1.5 mg / mL) and 2.5 mL of deionized water; (iii) the particles were centrifuged at 15,000 rpm for 15 minutes at 10°C; (iv) the concentration of ICC in the supernatant was determined to ascertain the mass of uncaptured ICG; and (v) the mass, EE%, and ICG% of the captured ICG were calculated. Table 4 shows the results of ICG encapsulation in the alginate / chitosan oligosaccharide particles.
[0095]
[0096] Figure 20 This paper compares the encapsulation efficiency (EE%) and loading percentage of ICG functionalization in biodegradable chitosan oligosaccharide / alginate colloidal particles using two different methods. It is important to emphasize that although a higher encapsulation efficiency percentage and captured ICG mass were achieved in the first method (i), the release kinetics in the samples prepared by the two methods are likely to be very different. Since ICG may adsorb on the surface, release is expected to be much faster in the sample prepared by the first method (i), while release is likely to be slower in the sample loaded by the second method (ii), where ICG is expected to reside in more of the internal region of the particle.
[0097] Based on the foregoing, it can be seen that systems for releasing bioactive compounds comprising biodegradable colloidal particles have been designed for application in the pharmaceutical and biotechnology industries. It will be apparent to any expert in the art that the embodiments of the invention shown above and in the accompanying drawings are merely illustrative and not intended to limit the invention, as many significant changes in detail are possible without departing from the scope of the invention. For example, the methods described above can be used to obtain biodegradable colloidal particles, and different biopolymers can be used from those shown in the previously described embodiments.
[0098] Therefore, the invention should not be considered limited except as required by the prior art and the scope of the appended claims.
Claims
1. A system for releasing a bioactive compound, characterized in that, The system comprises biodegradable colloidal particles, which in turn contain anionic polysaccharides, cationic polysaccharides, and at least one bioactive compound; the average size of the biodegradable colloidal particles is less than 500 nm.
2. The system according to claim 1, wherein, The anionic polysaccharide is alginate.
3. The system according to claim 1, wherein, The cationic polysaccharide is selected from chitosan, chitosan oligosaccharide, or a mixture thereof.
4. The system according to claim 3, wherein, The cationic polysaccharide is chitosan oligosaccharide.
5. The system according to claim 1, wherein, The bioactive compound has antitumor activity and is photosensitizing.
6. A method for obtaining biodegradable colloidal particles, characterized in that, Includes the following steps: (a) Estimate the stoichiometric charge ratio between anionic and cationic polysaccharides based on the degree of deesterification of anionic polysaccharides and the degree of deacetylation of cationic polysaccharides; (b) Prepare a solution containing anionic polysaccharides based on the stoichiometric charge ratio and adjust it to an acidic pH. (c) Prepare a solution containing cationic polysaccharides according to the stoichiometric charge ratio and adjust it to an acidic pH; (d) Adding the solution containing cationic polysaccharides to the solution containing anionic polysaccharides to obtain biodegradable colloidal particles; (e) reducing the particle size of the biodegradable colloidal particles; (f) separating the biodegradable colloidal particles from the supernatant; and (g) resuspending the biodegradable colloidal particles and storing them.
7. The method according to claim 6, wherein, The anionic polysaccharide is alginate.
8. The method according to claim 6, wherein, The cationic polysaccharide is selected from chitosan, chitosan oligosaccharide, or a mixture thereof.
9. The method according to claim 8, wherein, The cationic polysaccharide is chitosan oligosaccharide.
10. The method according to claim 6, wherein, The solvent used to prepare the polysaccharide solution is water.
11. The method according to claim 6, wherein, The degree of deesterification in step (a) is determined by potentiometric titration or conductivity titration.
12. The method according to claim 6, wherein, The degree of deacetylation in step (a) is determined by nuclear magnetic resonance (NMR), ultraviolet-visible spectrophotometry, potentiometric titration or conductivity titration or FTIR-ATR spectrophotometry.
13. The method according to claim 6, wherein, The pH of the solution of the anionic polysaccharide was adjusted to 4.
14. The method according to claim 6, wherein, The pH of the solution of the cationic polysaccharide was adjusted to 5.
15. The method according to claim 6, wherein, The solution of the cationic polysaccharide is filtered before being used in step (d).
16. The method according to claim 6, wherein, Step (d) is carried out by continuous dripping with vigorous stirring.
17. The method according to claim 6, wherein, The particle size reduction in step (e) is carried out by ultrasonic treatment of the biodegradable colloidal particles: the particles are placed in a container in ice water to relieve overheating for 5 to 10 minutes.
18. The method according to claim 6, wherein, The separation in step (f) was carried out by centrifugation at a speed of 6000 rpm to 7000 rpm.
19. The method according to claim 6, wherein, The resuspension in step (g) is carried out by vortexing for at least 10 minutes.
20. The method according to claim 6, wherein, The method includes an additional step of functionalizing the biodegradable colloidal particles with at least one bioactive compound.
21. The method according to claim 20, wherein, The functionalization step is performed prior to step (d), wherein the solution of the cationic polysaccharide obtained in step (e) is mixed with a solution containing at least one bioactive compound.
22. The method according to claim 20, wherein, The functionalization step is performed after step (g) and includes the following steps: (i) adding a solution containing at least one bioactive compound to a container containing the biodegradable colloidal particles; (ii) adding deionized water to the mixture; (iii) stirring; (iv) centrifuging; (v) resuspending the previously functionalized biodegradable colloidal particles in deionized water to remove uncaptured bioactive compounds; and (vi) freeze-drying the functionalized biodegradable colloidal particles.
23. The method according to claim 22, wherein, The stirring in step (iii) is carried out on a vortex oscillator for at least 1 hour.
24. The method according to claim 22, wherein, The centrifugation in step (iv) is performed at a temperature of at least 10 °C at 9,000 to 15,000 rpm for 15 to 30 minutes.
25. The method according to claim 22, wherein, The bioactive compound has antitumor activity and is a photosensitizer.