Albuminated coated liposomes (Machine-translation by Google Translate, not legally binding)

专利摘要:
Liposomes coated with albumin. The present invention relates to the preparation of particles by micro-encapsulation with albumin of liposomes of different composition and zeta potential in the absence of organic solvents and without the need for extreme conditions of temperature and/or pressure, useful as carriers of active principles with potential interest in the field of medicine. (Machine-translation by Google Translate, not legally binding)
公开号:ES2596558A1
申请号:ES201530974
申请日:2015-07-07
公开日:2017-01-10
发明作者:María José DE JESÚS VALLE；Amparo SÁNCHEZ NAVARRO
申请人:Universidad de Salamanca；
IPC主号:

专利说明:

5
10
fifteen
twenty
25
30
35
LIPOSOMES COVERED WITH ALBUMINE DESCRIPTION
The present invention relates to spherical particles constituted by albumin coated liposomes and their method of obtaining based on flocculation induced by electrostatic attraction. The resulting particles can encapsulate a wide range of active ingredients or molecules, making them a useful system as a versatile, biocompatible and biodegradable carrier for the controlled release of drugs and / or diagnostic agents.
STATE OF THE TECHNIQUE
With the emergence of nanotechnology and the great advances in biomaterial sciences, controlled release systems of increasingly sophisticated drugs that pursue diverse objectives are proposed, including vectorization along with the selective release of the active substance in response to biological signals.
Thus, multifunctional formulations that combine different materials such as lipids, proteins, oligosaccharides and synthetic polymers are a continuous object of study. In this sense, liposomes are currently one of the most promising systems in this field and numerous strategies based on the selection of the components of the lipid bilayer are being tested to achieve controlled and restricted biophase release (Qian et al. Journal of Controlled Release, 2015, 207 {10), 86-92; Movahedi et al. Nanomedicine, 2015, 11 (6), 1575-1584), as well as for vaccine formulation.
On the other hand, because the stability of the liposomes in the bloodstream is low due to their interaction with the different plasma components, stabilization strategies are sought, with pegylation being the most applied so far, although it is also true that the molecules of PEG can accumulate in some cells altering their functions (Romberg et al., Bioconjugate Chem., 2005, 16 (4), 767-774). For this reason it is about replacing PEG with other products such as albumin, heparin, chitosan or hyaluronic acid.
5
10
fifteen
twenty
25
30
35
Albumin, a hydrophilic molecule with an isoelectric point of approximately 5, is the most abundant plasma protein in mammals, with important physiological functions in the regulation of colloid osmotic pressure and the transport of numerous solutes (fatty acids, hormones, bile acids , amino acids, metals, etc.) from the bloodstream to the tissues. In addition, it seems that albumin facilitates the endothelial transcytosis of certain plasma components due to its binding to a membrane receptor (albondine) with the consequent formation of vesicles called caveolae. On the other hand, it seems that albumin is involved in the selective tissue distribution of certain drugs to which it protects against oxidation phenomena, while influencing their kinetic profile (Feng et al., Int. J. Mol. Sci. 2014, 15 (3), 3580-3595; Mita et al., Invest New Drugs, 2015, 33, 341-348).
These properties, together with its greater uptake and accumulation in tumor tissues, inflamed and infected, makes albumin a good candidate to be included in formulations that seek a selective release in the tissues affected by the aforementioned processes, especially if they have low aqueous solubility ( Krazt et al., J. Control. Release, 2008, 132 (3), 171-183).
In this sense, bovine albumine nanoparticles have proven effective in the incorporation of certain dyes, drugs and vectorizing agents. For antineoplastic drugs, albumin is presented as an excellent component of the formulation since it is a product with an adequate molecular weight to benefit from the EPRE (enhanced permeability and retention effect) described in tumor tissues. The first drug marketed in oncology based on albumin nanoparticles was Abraxane® with indication for the treatment of breast cancer; It consists of 130nm nanoparticles consisting of human albumin and paclitaxel. This patented technology as nab-technology promises a wide application for other drugs similar to paclitaxel. Currently, a tyrosine kinase inhibitor approved for use, together with capecitabine, is studied for docetaxel and lapatinib for the treatment of cancer in patients with HER2 epidermal growth receptor expression. Lapatinib incorporated into albumin particles obtained with nab-technology has demonstrated superior antitumor efficacy to the currently marketed formulation (Wan et al., International Journal of Pharmaceutics, 2015, 484 (1-2), 16-28). The incorporation of doxorubicin and tacrolimus into nanoparticles of
5
10
fifteen
twenty
25
30
35
albumin; in the first case, by means of a simple co-precipitation procedure, particles are released that release more antineoplastic in the tumor by a passive vectorization mechanism. For tacrolimus the procedure applied is more complex and involves the formation of emulsions and their subsequent homogenization by high pressures (Zhao et al., Pharmaceutical Nanotechnology, 2015, 483 (1-2), 180-187), but it manages to significantly reduce the nephrotoxicity of the product.
As regards the combination of liposomes and albumin, so far this strategy has been fundamentally explored in the field of immunology, using albumin as an adjuvant. The strategies described in the literature resemble pegylation because they are based on the covalent binding of albumin to some component of the liposome bilayer, for which it is necessary to modify the molecular structure of albumin. Thus, thiolated bovine albumin covalently binds to the butyryl phosphatidylethanolamine which is part of the lipid vesicles and the resulting product demonstrated high efficacy to enhance the immune response. When the immunological response to thiolated ovalbumin covalently bound to liposomes containing doxorubicin was evaluated, what was observed with empty liposomes did not occur because doxorubicin inhibits the activity of phagocytic cells in the liver. This confirms that modified albumin is not inert and triggers unwanted immune processes if it is intended to be used as an excipient of non-vaccine formulations. There is hardly any data on the combination of albumin and liposomes for drug formulation and none uses native, unmodified albumin; Published studies refer to the use of denatured albumin to coat pegylated or non-pegylated liposomes containing doxorubicin or the G3139 antisense oligonucleotide, and both agree to affirm the advantages of incorporating albumin into the formulation.
According to the above, so far no liposome coating methods have been described or applied with unmodified albumin by an induced flocculation phenomenon, achieving a versatile, biocompatible, biodegradable and harmless pharmaceutical vehicle. This document also describes the method of preparing these particles consisting of liposomes encapsulated with albumin, based on the induction of flocculation phenomena, which takes place in the absence of organic solvents, at atmospheric pressure and without high temperatures.
5
10
fifteen
twenty
25
30
35
DESCRIPTION OF THE INVENTION
Spherical particles have been designed, formulated and characterized that comprise at least one liposome, of different composition and / or zeta potential, coated with albumin, capable of containing different active ingredients, or other molecules or products. Its characteristics vary according to the experimental conditions applied during the production process, which is carried out in the absence of organic solvents and without the need for extreme temperature and / or pressure conditions, although this procedure is not limiting and could also be applied to liposomes obtained by any other method.
Products without active ingredient and loaded with different active ingredients were prepared, such as vancomycin and ciprofloxacin. The first, a glycopeptide of complex structure with bactericidal effect, was selected as a water soluble drug model with moderate affinity for albumin and the kinetic profile of vancomycin release was studied in vitro by dialysis assays. The second, an antibiotic from the quinolone group, with physicochemical characteristics very different from vancomycin in terms of molecular weight (331.34 g / mol for ciprofloxacin versus 1449.25 g / mol for vancomycin) and acid-base character (pKa = 6.03 for ciprofloxacin versus vancomycin diprotic character with pKa = 2.99 and pKa = 9.93), spherical particles are also incorporated into the liposomes and form the size, zeta potential and active substance content They depend on the experimental conditions in which the process is performed.
According to the results obtained, it can be affirmed that the proposed particles constitute biocompatible, biodegradable and harmless transport vehicles, and of great versatility in terms of size, zeta potential and content in active principles, which gives them a high potential for formulation of therapeutic agents, diagnostic agents and vaccines.
Therefore, a first aspect of the present invention relates to a spherical particle formed by an albumin coated core by electrostatic interaction, wherein said core comprises at least one liposome that can be anionic or cationic zeta potential between + 90mV and - 90mV
5
10
fifteen
twenty
25
30
35
By the term "liposome" is meant a spherical vesicle in which an aqueous nucleus and at least one membrane composed of a double layer of amphiphilic lipids, also called lipid bilayer, which includes hydrophilic and lipophilic parts are distinguished.
The liposomes can be single, oligo- or multilamellar, that is, they can comprise one or more double layers of lipids. Although those of the invention, the most suitable and preferred for topical application are unilamellar.
The term "albumin" is generally understood as a native or recombinant mammalian albumin. More preferably, the albumin is of human, bovine (BSA), murine, or rabbit origin; Other possible albumins are ovalbumin and lactalbumin. Even more preferably, the albumin used is recombinant human serum albumin, or fresh or lyophilized bovine serum albumin.
In a preferred embodiment the particle has a micrometer size with a particle diameter between 0.1 ^ m and 100 ^ m, preferably between 0.2 ^ m and 1 ^ m.
In a preferred embodiment, the particle has a nanometric size with a particle diameter between 0.1nm and 100nm.
In another preferred embodiment, the albumin is unmodified albumin of human origin.
In another preferred embodiment the lipid bilayer constituents of the liposome include: a) phospholipids, both natural and synthetic origin such as phosphatidylcholine, or cholesterol and b) another lipid with ionic charge, that is, positively or negatively charged; or any of its mixtures.
Among the preferred ionic charged lipids are, for example, dimethyldioctadecylammonium, linoleic acid and phostatidylglycerol, among others.
In another aspect of the invention, the spherical particle comprises a molecule or an active ingredient encapsulated in the liposome, or trapped in the albumin sheath, or encapsulated in the liposome and trapped in the albumin sheath.
5
10
fifteen
twenty
25
30
35
As used herein, the term "active ingredient" means any component that potentially provides a pharmacological activity or other effect different in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body. of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.
Preferred active ingredients are selected from the following non-limiting list of: antifungals (amphotericin, fluorocytosine, itraconazole, posaconazole, caspofungin, anidulafungin, micafungin, isavuconazole, aminocandin, among others), antibiotics (ciprofloxacin, vancomycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin, erythromycin , clarithomycin, penicillins and cephalosporins, rifampicin, isoniazid and streptomycin, quinolones, among others), progestogens (ethinyl estradiol, norelgestromin, levonorgestrel, drospirenone, norgestimate, among others), antineoplastic drugs (doxorubicin, pactinothrometin, pactinothrombin, cactin , tegafur, methotrexate, among others), antiretrovirals: (efavirenz, atazanavir, sofosbuvir, daclastavir, ledipasvir, ombitasvir, among others), analgesics and anti-inflammatories (for example non-steroidal anti-inflammatories, triptans (for example eletriptan or almotriptan)) cyclooxygenase 2 (for example celecoxib), derivatives of oxicam (for example piroxicam) pyrazolic derivatives (for example phenylbutazone), diagnostic agents, paramagnetic material, etc.
In a preferred embodiment, compounds loaded with vancomycin and ciprofloxacin were prepared.
Another aspect of the invention relates to a composition comprising the spherical particle of the invention, this composition being preferably pharmaceutical or cosmetic. In a preferred embodiment of this aspect of the invention, the pharmaceutical or cosmetic composition further comprises pharmaceutically or cosmetically acceptable excipients.
The term "excipient" refers to a substance that helps the administration of any of the components of the product of the invention, stabilizes said components or aids in the preparation of the pharmaceutical or cosmetic composition in the sense of giving it consistency and thus stabilizing the suspension. So, the
5
10
fifteen
twenty
25
30
35
Excipients could have the function of keeping the components together such as starches, sugars or cellulose, smell or deodorize function, dye function, drug protection function, lyophilization function of the drug or cosmetic, among others. Therefore, the term “excipient” is defined as that matter that, included in the galenic forms, is added to the active principles or to their associations to help its administration, penetration into the skin or pimples, enable its preparation or formulation and stabilize it , modify its organoleptic properties or determine the physicochemical properties of the pharmaceutical composition and its bioavailability. The "cosmetically or pharmaceutically acceptable" excipient must allow the activity of the compounds of the composition, that is, to be compatible with said components. Examples of excipients are isotonizers, pH regulators, binders, fillers, disintegrators, lubricants, flavorings or aromas and dyes. More specific non-limiting examples of pharmaceutically or cosmetically acceptable excipients are starches, sugars, hyaluronic acid, xanthan, glycerol, xylitol, sorbitol, or glycerin among others.
In addition, the composition may comprise various antioxidants, such as vitamin E, vitamin A and vitamin C, which would primarily provide a chemical stabilizing effect of the composition.
Another aspect of the invention relates to the use of the particles of the invention as a transport vehicle, preferably for the controlled release of drugs and particularly non-water soluble active ingredients.
Another aspect of the invention relates to the use of the particles of the invention for the preparation of a medicament, therapeutic products or vaccines, preferably for the treatment of cancer, HIV and Hepatitis C.
Likewise, thanks to the fact that the described compounds have three differentiated deposits: aqueous liposome nucleus, lipid bilayer of liposomes and protein with binding and transport capacity, each one capable of harboring active principles of different solubility and affinity, which It is interesting for the development of a drug for the treatment in combination therapies.
5
10
fifteen
twenty
25
30
In addition, the vehicle described in the present invention could be applied to the formulation of medicaments that can be applied according to the following, non-limiting list:
- Oral administration: for drugs with low aqueous solubility for which the conventional formulation requires working with organic solvents and / or incorporating surfactants and other non-harmless solubilizing excipients.
- Trans-mucosal administration: the interaction of albumin with the mucin present in the mucous secretions favors the mucoadhesion and prolonged cession of the drug, which presents special interest for the routes: pulmonary, for passive vectorization of drugs whose biophase is located in the respiratory system (antibiotics, antineoplastic, anti-inflammatory); buccal, for passive vectorization of drugs whose biophase is located in the oral cavity (antibiotics, antifungals, antineoplastic drugs, anti-inflammatories); nasal, for vaccines; vaginal, for the treatment and prophylaxis of vaginal infections, (antibiotics, antifungals, antineoplastic, anti-inflammatory); rectal (antibiotics, antifungals, antineoplastic, anti-inflammatory); eye (antibiotics, antifungals, anti-inflammatories).
Transdermal
- Parenteral routes: size requirements are achieved by controlling the conditions of the process, specifically the zeta potential of the gallbladder and the amount of albumin incorporated into the compound. Within the parenteral routes, the following stand out: intravenous, especially for antineoplastic drugs and antibiotics due to the ability of albumin to accumulate in infected tumors and tissues; intramuscular, intradermal and subcutaneous (vaccines and others).
Another aspect of the invention relates to a method of obtaining the particles of the invention which comprises contacting a liposome suspension with an albumin solution by flocculation at a pH of between 4 and 8, according to the net liposome load.
In a preferred embodiment, at a fixed volume of the liposome suspension, in water or buffer solution, the same volume of albumin solution, in water or buffer solution, of concentration between 0.05 and 30% is added. In a more preferred embodiment the concentration of albumin is 1%.
5
10
fifteen
twenty
25
30
In another preferred embodiment, the flocculation stage is induced at pH between 3 and 5 when starting from liposomes with negative zeta potential and is induced at pH between 5 and 8 when starting from liposomes with positive zeta potential.
In another preferred embodiment, the buffer solution preferably comprises phosphate or citrate salts.
In another preferred embodiment, the transport vehicles obtained by the described method were coated with bovine albumin of human origin by a phenomenon of flocculation in aqueous medium, atmospheric pressure (approximately 1 atmosphere) and temperature between 2 and 10 ° C. In a more preferred embodiment this flocculation temperature is 4 ° C.
Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1-4 Morphology of albumin coated liposomes obtained by scanning electron microscopy (SEM) of different sizes.
FIG. 5. In vitro release kinetics of vancomycin. Results of studies conducted with compounds prepared from cationic liposomes. The amounts ceded (milligrams) at different times for a period of 48 h are illustrated.
FIG. 6. Analysis of the curves of remaining amounts in compounds. It shows that the kinetic process of drug release (vancomycin) is poly-exponential in all cases, regardless of the amount of albumin and active ingredient incorporated.
5
10
fifteen
twenty
25
30
35
EXAMPLES
The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
Example 1
Preparation of lioosomes
The constituent lipids of the lipid bilayer of the liposome are mixed with Milli- Q water previously heated to 60 ° C, stirring the mixture until dispersion of the lipids and then placed in an ultrasonic bath (50 Hz) for 20 min at 60 ± 2 ° C. The liposomes, whose composition includes egg phosphatidylcholine, cholesterol and an ionic-charged lipid (dimethyldioctadecylammonium, linoleic acid or phostatidylglycerol), were prepared in any case from the same concentration of lipids in water (1.73% w / v) .
The direct sonication of the mixture of lipids and water makes it possible to obtain liposomes without using organic solvents, as demonstrated in previous work done with liposomes without ionic lipids. The cationic and anionic liposomes prepared had mean values of hydrodynamic diameter of 56.98 nm (PDI = 0.29) for cationics and 50.84 nm (PDI = 0.24) for anionics. The zeta potential was found to be +61.9 ± 2.08mV and -47, 95 ± 4.71mV, respectively.
Example 2
Preparation of liposomes loaded with active substance
To obtain liposomes loaded with vancomycin, a solution of the active substance (vancomycin or ciprofloxacin) with a concentration of 5 mg / mL was used. The sonicated samples were kept 1 h at rest, at room temperature for 60 min, so that the process of lipid assembly and vesicle formation is completed; They were then filtered through 0.22 ^ m membrane. An aliquot of the liposome suspension was taken for its characterization (morphology, size, zeta potential and content in active principle) and the rest was used to cover it with albumin for the formation of the transport vehicles.
5
10
fifteen
twenty
25
30
35
The efficacy of active substance uptake in liposomes was 15% in cationics and slightly lower in anionics in the case of vancomycin.
Example 3
Preparation of the encapsulated liposomes
Lipid vesicles of different composition and zeta potential, obtained by the described method, were coated with bovine albumin. The same volume of albumin solutions of different concentration (0.1-3%) was added dropwise to a fixed volume of the liposome suspension. A phenomenon of electrostatic attraction flocculation was induced between liposomes and albumin, which requires the pH adjustment to a value below the isoelectric point (pH = 5) when starting from liposomes with negative zeta potential. The resulting samples were maintained with mechanical stirring at room temperature for 15 min and subsequently in a 4 ° C incubation bath and gentle stirring for 20 h; After this time the samples were centrifuged at 10,000 rpm and 4 ° C and the pellet supernatant was removed. The volume of supernatant was measured, albumin and vancomycin levels were quantified and the presence of liposomes was investigated. An aliquot was separated from the pellet to characterize the particles formed (morphology, size, zeta potential, and drug content) and the rest was destined for the in vitro vancomycin assignment test.
The centrifugation of the flocculated samples allowed the pellet to be separated without difficulty from the supernatant; When the latter was analyzed by microscopy and DLS, no liposomes were found and the quantification analysis of albumin and vancomycin showed that a small fraction remains in the supernatant, the rest being incorporated into the particles formed.
Example 4
Characterization of liposomes and encapsulated liposomes
Morphology
The liposome suspensions and the pellets obtained were observed under a microscope using optical microscopy equipment connected to a camera and image capture software (Canon remote capture) and also with microscopy equipment.
5
10
fifteen
twenty
25
30
35
scanning electronics (SEM), for which the samples were previously subjected to a fixing process using poly-L lysine and osmium as fixing agents.
Regarding the morphology of the vehicles obtained, they have a spherical shape and smooth surface (Fig. 1-4). In addition, it was observed that the size increases as the amount of albumin added increases, regardless of whether the starting liposomes are anionic or cationic.
Zeta size and potential
The hydrodynamic diameter (dh) and polydispersion index (PDI) of the particles were determined before (liposomes) and after coating with albumin (encapsulated liposome) using two devices, Mastersizer 2000, which analyzes particles larger than 0.5 pm and another Zetasizer nano ZN, which detects and analyzes the smaller ones. The determinations were made at 25 ° C, with a correlation function for an angle of 173 ° in the Zetasizer nano and 90 ° in the Mastersizer, applying the Stokes-Einstein ratio for the dh determination. The samples were diluted with Milli-Q water until the optimum degree of oscillation was obtained for the determinations. The intensity and number distribution curves were recorded and analyzed to estimate the value of dh and PDI. Likewise, the zeta potential of both the starting liposome particles and the vehicles obtained was measured using the Zetasizer nano equipment and applying the M3-PALS technique that combines "Doppler Laser Velocimetry" and "Phase Analysis Light Scattering-PALS".
It is observed that the zeta potential of the resulting transport vehicles takes different values that depend on the initial load of the liposomes and the amount of albumin incorporated therein. For cationic liposomes the proportion of incorporated albumin decreases slightly as the amount added (EEalb = 9.2 ± 1.05%, 9.02 ± 1.82% and 8.9 + 0.97), although Differences did not show statistical significance (p> 0.05). For these, the effectiveness of active ingredient incorporation progressively increased as the amount of albumin incorporated into the vehicles increased (EEvan = 37.87 ± 8.01%, 45.9 ± 4.24% and 52.0 ± 2.28 %, respectively), presenting the statistical significance difference (p <0.05).
As can be seen from the values shown in the following table, for the anionic liposomes neither EEalb nor EEvan were found to be dependent on the amount of albumin
added, at least with the concentrations of protein and zeta potential tested. In this case, EEvan, in addition to not changing with albumin, has higher values than those corresponding to cationic liposomes (64.00 ± 1.65 vs. 52.06 ± 2.28).
 Liposome load  Albumin EEalb (%) EEvan (%) Albumin (mg) Vancomycin (mg)
 +  1% 9.22 ± 1.05 37.88 ± 8.01 5.52 ± 0.55 11.36 ± 2.40
 +  2% 9.08 ± 1.82 45.90 ± 4.24 10.9.2 ± 2.18 13.77 ± 1.27
 +  3% 8.98 ± 0.97 52.06 ± 2.28 16.2 ± 1.46 15.61 ± 1.07
 -  1% 8.8.0 ± 0.62 64.00i1.65 5.28 ± 0.32 19.20 ± 0.36
 -  2% 8.87 ± 0.32 63.08 ± 0.43 10.64 ± 0.31 18.90 ± 0.76
 -  3% 8.81 ± 0.64 63.00i1.31 15.78 ± 0.67 18.90 ± 0.24
5
Active substance content of liposomes
A fixed volume (V) of the liposome suspension is placed in a dialysis bag (cut-off 12-14k Da) that is suspended in 25 ml_ of Milli-Q water previously heated 10 to 37 ° C, in a closed tube submitted Stirring Samples are taken from the external medium, every 15 min, replenishing the volumes removed with the same amount of fresh medium, until dialysis equilibrium is reached. Parallel tests were performed under identical conditions but including the same volume (V) of a vancomycin solution of known concentration (5 mg / ml_) in the dialysis bag, as a reference. The amount of vancomycin in the liposomes (Qlip) was determined from the equilibrium concentrations of active principle in the dialysate from the reference and the liposomes (CeryCelip, respectively).
Cer = f (C x V) / 25 + V
Celip = f (C x (V-Vlip)) 125 x (V - VIip)
20 Qlip (mg) = Vlip x C
Where f is the fraction of active ingredient available for the exchange of dialysis, C and V are the concentration of drug and the volume within the dialysis bag, respectively and Vlip is the volume trapped in the liposomes. The effectiveness of
5
10
fifteen
twenty
25
30
35
Encapsulation of active ingredient in liposomes (EElip) was expressed as 100 x (Qlip / Qi), where Qi is the initial amount of vancomycin used for the preparation of liposomes.
Composition of the microspheres with encapsulated liposomes The composition of the particles formed by the interaction between liposomes and albumin, induced by the applied experimental conditions, was determined indirectly from the composition of the supernatant separated by centrifugation of the flocculated samples. Since centrifugation allows the pellet to be perfectly separated from the supernatant, it is assumed that all components of the mixture (liposomes, albumin and vancomycin) that are not found in the supernatant are the pellet (encapsulated liposome). Accordingly, the amount of albumin and vancomycin trapped in the encapsulated liposomes (Qalb and Qvan, respectively) was calculated as follows:
Qalb (mg) = (Qalb) i - (Vs x Calb)
Qvan (mg) = (Qvan) i - (Vs x Cvan)
Where (Qalb) i and (Qvan) i are the amounts of protein and active ingredient initially added to the flocculated samples, respectively; Vs the measured volume of supernatant and Calb and Cvan the concentrations of albumin and vancomycin measured in the supernatant, respectively.
The efficacy of encapsulation of albumin and vancomycin in liposomes (EEalb EEvan, respectively) was expressed as the percentage trapped of each product in the particles.
EEalb (%) = 100 Qalb / (Qalb) i EEvan (%) = 100 Qvan / (Qvan) i
Example 5
Kinetics of release of the active substance from the liposomes
The vancomycin assignment of encapsulated liposomes was characterized in vitro by a dialysis assay, similar to that applied for the determination of the active substance content of liposomes. The pellet separated by centrifugation was dispersed in 1.5 mL of Milli-Q water and included in a dialysis bag (cut-off 12-14k Da) which, in turn, was suspended in 25 mL of Milli-Q water previously heated to 37 ° C in
5
10
fifteen
twenty
25
30
35
tube closed and subjected to stirring throughout the test. At previously scheduled times, samples were taken from the external medium, replenishing with the same volume of fresh medium at the same temperature. The concentrations in the samples were measured and the amounts of active ingredient assigned at different times (Q) tn were calculated as follows:
(Q) tn = (Ctn / f) x 25 + Z (Ctn-1 x 2) / f
Where Ctn is the concentration of active ingredient in the dialysate at time tn, Y (Ctn-1 x 2) is the amount of drug withdrawn in the previous samples and f is the fraction of active ingredient that is exchanged through the dialysis membrane .
The curves of remaining amounts of vancomycin in encapsulated liposomes (Qr) tn are obtained from the differences between the amount trapped in the liposomes and the amounts assigned:
(Qr) tn = Qvan- (Q) tn
These curves were analyzed and adjusted to the following poly-exponential equation using a non-linear regression program (Winnonlin) based on the calculation of coefficients (Ai) and exponents (Ri), considering the AKAIKE criteria for the selection of the best fit of the curves :
(Qr) tn = Y A¡ e-Rit
The entire experimental procedure described (from the preparation of liposomes to the kinetics of in vitro assignment) was performed in quadruplicate and the data presented are the average values of four replicates.
The results of studies carried out with vehicles prepared from cationic liposomes are shown in Fig. 5, which illustrates the amounts assigned at different times for a period of 48 hours.
Differences are observed between the total amounts ceded by vehicles with a lower amount of albumin (Q48h = 6.57 ± 1.00 mg and EEvan = 37.87 ± 8.01%) and those of the others with higher EEalb (Q48h = 7.89 ± 0.38 mg and 7.99 ± 0.50 mg for EEvan = 45.9 ± 4.24% and 52.0 ± 2.28%, respectively), statistical significance (p <0.05) . In none of the cases the release of all the active substance content occurs, despite reaching the asymptotic value for the three conditions. This leads to thinking
5
10
fifteen
twenty
25
30
that the amount of vancomycin trapped in the liposomes does not yield under the in vitro conditions of the assay, which is consistent with the biopharmaceutical behavior of the lipid vesicles, which release their content when interacting with specific tissues or cells. Taking into account that the undisputed amount is progressively greater as the amount of albumin increases, it is felt that the number of trapped vesicles grows with increasing albumin.
The analysis of the curves of remaining quantities in the transport vehicles obtained, shown in Fig. 6, demonstrates that the kinetic process of release of the active substance is of a poly-exponential nature in all cases, regardless of the amount of albumin and active ingredient ( vancomycin) incorporated into the compounds.
The results of the analysis of the average curves show a three-phase kinetic profile with slightly higher exponent values for the compounds with lower albumin content and active principle, which means slightly faster release rates, probably due to the smaller size (larger specific surface ) of particles with less protein content and active substance. The following table shows the results of the linear regression analysis performed with the remaining quantity curves and shows the coefficient and exponent values obtained for the different experimental conditions tested.
 Liposome load  Albumin Conc A1 A2 A3 R1 R2 R3 AIC
 +  1% 3.53 2.93 5.91 1.18 0.36 0.0049 69.80
 +  2% 3.90 4,015 6.90 1.04 0.30 0.0037 48.24
 +  3% 2.43 4.95 9.18 1.03 0.32 0.0042 35.49
Example 6
Quantification of vancomycin and albumin in the samples
In the samples from the dialysis test, vancomycin is quantified directly since the dialysis membrane used has a cut-off of 12-14 kDa, which allows the exchange of vancomycin (pm = 1500 Da) but not albumin (pm = 77 kDa). This type of sample only contains water and vancomycin, which allows its quantification from the absorbance measured by UV spectroscopy
at a wavelength of 220nm. Patterns of the active substance were prepared in Milli-Q water in a concentration range of 0.01 mg / ml to 0.1 mg / ml and 0.1 mg / ml to 0.25 mg / ml_.
5 In the supernatant samples obtained by centrifugation of the flocculates there is vancomycin and albumin, the latter interfering with the vancomycin quantification test described above. In order to remove the albumin from the sample, the supernatants were super-centrifuged with membrane tubes ((Centrisart-1, 10,000Da) that provide an albumin-free ultrafiltrate or any other solute of pm> 12-14 kDa Vancomycin was quantified in the utrafiltrates obtained under the same conditions described above.
For the quantification of albumin in the supernatants a technique of UV spectroscopy Aemission = 200nm was also used. Albumin standards were prepared in Milli-Q water in a concentration range of 0.05-0.20 mg / ml. In this case the presence of vancomycin does not affect the absorbance of the protein, so no prior treatment of the sample is required.
twenty

权利要求:
Claims (29)
[1]
5
10
fifteen
twenty
25
30
35
1. - Spherical particle formed by an albumin coated core by electrostatic interaction, where said core comprises at least one liposome, anionic or cationic, of zeta potential between + 90mV and - 90mV.
[2]
2. - Particle according to claim 1, wherein the particle is micrometric in size with a particle diameter between 0.1 ^ m and 100 ^ m.
[3]
3. - Particle according to claim 2, wherein the particle is micrometric in size with a particle diameter between 0.2 ^ m and 1 ^ m.
[4]
4. - Particle according to claim 1, wherein the particle has a nanometric size with a particle diameter between 0.1 nm and 100 nm.
[5]
5. - Particle according to any of the preceding claims, wherein the liposome comprises phosphatidylcholine or cholesterol, and a lipid with ionic charge, or any of its mixtures.
[6]
6. - Particle according to any of the preceding claims, wherein the lipid with ionic charge is selected from dimethyldioctadecylammonium, linoleic acid or phostatidylglycerol.
[7]
7. - Particle according to any of the preceding claims, wherein the albumin is unmodified albumin of human or bovine origin, fresh or lyophilized.
[8]
8. - Particle according to claim 7, wherein the albumin is unmodified albumin of human origin.
[9]
9. Particle according to any of the preceding claims which further comprises an active ingredient.
[10]
10. Particle according to claim 9, wherein active ingredient is encapsulated in the liposome, trapped in the albumin sheath or encapsulated in the liposome and trapped in the albumin sheath.
5
10
fifteen
twenty
25
30
35
[11]
11. Particle according to claim 10, wherein the active ingredient is selected from the list comprising antifungals, antibiotics, progestogens, antineoplastic, antiretroviral, analgesic or anti-inflammatory, diagnostic agents or paramagnetic material.
[12]
12. Particle according to claim 10, wherein the active ingredient is selected from the list comprising vancomycin, ciprofloxacin, amphotericin B, fluorocytosine, itraconazole, posaconazole, caspofungin, anidulafungin, micafungin, isavuconazole, aminocandin, erythromycin, azithromycin, claromycinin, clarithycinin, clarithycinin, clarithycinin, clarithycinin, clarithycinin, clarithycinin, clarithycinin, clarithycinin, penicillin cephalosporins, rifampicin, isoniazid, streptomycin, quinolone, ethinylestradiol, norelgestromin, levonorgestrel, drospirenone, norgestimate, doxorubicin, epirrubcina, dactinomycin, paclitaxel, docetaxel, lapatinib, capecitabine, tegafur, methotrexate, Efavirenz, atazanavir, sofosbuvir, daclastavir, ledipasvir, ombitasvir, NSAIDs, eletriptan, almotriptan, celecoxib, piroxicam, or phenylbutazone.
[13]
13. - Particle according to claim 12 wherein the active substance is vancomycin.
[14]
14. - Particle according to claim 12 wherein the active ingredient is ciprofloxacin.
[15]
15. - Composition comprising a particle according to claims 1 to 14, wherein said composition is preferably pharmaceutical or cosmetic.
[16]
16. - Use of the particle according to claims 1 to 14 as a transport vehicle, preferably for the controlled release of non-water soluble active ingredients.
[17]
17. - Use of the particle according to claim 16 as a vancomycin transport vehicle.
[18]
18. - Use of the particle according to claim 16 as a ciprofloxacin transport vehicle.
[19]
19. - Use of the particle according to claims 9 to 14 for the preparation of a medicament.
5
10
fifteen
twenty
25
30
35

[20]
20. - Use of the particle according to claims 9 to 14 for the preparation of teranostic products or vaccines.
[21]
21. - Use of the particle according to claims 9 to 14 for the preparation of a medicament for the treatment of cancer, HIV or Hepatitis C.
[22]
22. - Use of the particle according to claim 21 for the preparation of a medicament for the treatment in combination therapies.
[23]
23. - A method of obtaining the particle according to claims 1 to 14 which comprises contacting a liposome suspension with an albumin solution by flocculation at a pH between 4 and 8.
[24]
24. - A method of obtaining according to claim 23 in which a solution of albumin, in water or buffer solution, of concentration of between 0.05 and 30% is added to the liposome suspension, in water or buffer solution.
[25]
25. - A method of obtaining according to claim 24 wherein the buffer solution comprises phosphate or citrate salts.
[26]
26. - A method of obtaining according to claim 23 in which the flocculation stage is induced at pH between 3 and 5 when starting from liposomes with negative zeta potential.
[27]
27. - A method of obtaining according to claim 23 wherein the flocculation stage is induced at pH between 5 and 8 when starting from liposomes with positive zeta potential.
[28]
28. - A method of obtaining according to claims 23 to 27 wherein the reaction is carried out at a temperature between 2 and 10 ° C.
29 - A method of obtaining according to claim 28 wherein the flocculation temperature is 4 ° C.
[30]
30.- A process for obtaining the compound according to claims 23 to 29, in which an active ingredient is also added to the liposome suspension.

类似技术:

---

公开号 | 公开日 | 专利标题

---

Lim et al.2016|Polymeric nanoparticles in development for treatment of pulmonary infectious diseases

---

Dal Magro et al.2017|ApoE-modified solid lipid nanoparticles: A feasible strategy to cross the blood-brain barrier

---

ES2435944T3|2013-12-26|New formulations of pharmacological agents, methods for their preparation and methods for their use

---

Jain et al.2012|Gelatin coated hybrid lipid nanoparticles for oral delivery of amphotericin B

---

Luo et al.2015|Nanomedical engineering: shaping future nanomedicines

---

BRPI0908686B1|2020-09-29|METHOD OF PRODUCTION OF NON-IMMUNOGENIC NANOPARTICLES, THERAPEUTICALLY ACTIVE NON-IMMUNOGENIC NANOPARTICLE, THERAPEUTIC COMPOSITION, USE OF A NON-IMMUNOGENIC NANOPARTICLE AND KIT FOR THE PRODUCTION OF NON-IMMUNOGENIC NANOPARTICLES

---

G Nava-Arzaluz et al.2012|Single emulsion-solvent evaporation technique and modifications for the preparation of pharmaceutical polymeric nanoparticles

---

Kamboj et al.2013|Vesicular drug delivery systems: a novel approach for drug targeting

---

WO1996037232A1|1996-11-28|Stabilization of colloidal systems by the formation of ionic lipid-polysaccharide complexes

---

ES2385995B2|2013-05-21|NANOCAPPSULES WITH POLYMER COVER

---

Kumar et al.2020|A systematic study on chitosan-liposome based systems for biomedical applications

---

Yong et al.2016|Domino-like intercellular delivery of undecylenic acid-conjugated porous silicon nanoparticles for deep tumor penetration

---

Ribeiro et al.2017|Advances in hybrid polymer-based materials for sustained drug release

---

Pardeshi et al.2018|N, N, N‑trimethyl chitosan modified flaxseed oil based mucoadhesive neuronanoemulsions for direct nose to brain drug delivery

---

Molinaro et al.2020|Leukocyte-mimicking nanovesicles for effective doxorubicin delivery to treat breast cancer and melanoma

---

Gaber et al.2018|Protein-polysaccharide nanohybrids: Hybridization techniques and drug delivery applications

---

Miao et al.2018|Drug nanocrystals for cancer therapy

---

Xin et al.2017|PLGA nanoparticles introduction into mitoxantrone-loaded ultrasound-responsive liposomes: in vitro and in vivo investigations

---

Kamel et al.2019|Inhalable dual-targeted hybrid lipid nanocore–protein shell composites for combined delivery of genistein and all-trans retinoic acid to lung cancer cells

---

Lai et al.2020|Molecular design of layer-by-layer functionalized liposomes for oral drug delivery

---

Hamdi et al.2020|An integrated vitamin E-coated polymer hybrid nanoplatform: A lucrative option for an enhanced in vitro macrophage retention for an anti-hepatitis B therapeutic prospect

---

Gundloori et al.2019|Nanobased intravenous and transdermal drug delivery systems

---

García et al.2018|Self-assembled nanomaterials

---

Islan et al.2017|Development and tailoring of hybrid lipid nanocarriers

---

ES2596558B1|2018-01-26|LIPOSOMES COVERED WITH ALBUMIN

同族专利:

---

公开号 | 公开日

---

ES2596558B1|2018-01-26|

---

WO2017005853A1|2017-01-12|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

WO2004045583A1|2002-11-15|2004-06-03|Nipro Corporation|Liposome|

---

EP1655022A4|2003-08-01|2010-04-07|Nat Inst Of Advanced Ind Scien|Remedy or diagnostic for inflammatory disease containing target-directing liposome|US20200360362A1|2017-12-21|2020-11-19|Taiwan Liposome Co., Ltd|Sustained-release triptan compositions and method of use the same through subdermal route or the like|

法律状态:
2018-01-26| FG2A| Definitive protection|Ref document number: 2596558 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180126 |

优先权:

---

申请号 | 申请日 | 专利标题

---

ES201530974A|ES2596558B1|2015-07-07|2015-07-07|LIPOSOMES COVERED WITH ALBUMIN|ES201530974A| ES2596558B1|2015-07-07|2015-07-07|LIPOSOMES COVERED WITH ALBUMIN|

---

PCT/EP2016/066121| WO2017005853A1|2015-07-07|2016-07-07|Albumin-coated liposomes|