Method for producing liposomes

Abstract

This disclosure relates to a method for producing liposomes in order to achieve high encapsulation efficiency of encapsulated agents with a reduced number of production steps, notably avoiding the extrusion step of the classic liposomal production process. The liposomes of the invention are intended to carry a therapeutic agent such as an anticancer, antioxidant, anti-inflammatory, antipyretic, antibiotic, antiviral, antirheumatic, analgesic, growth factor, or mixtures thereof.

Description

METHOD FOR PRODUCING LIPOSOMES TECHNICAL DOMAIN

[0001] This disclosure relates to a method for producing liposomes in order to obtain high encapsulation efficiency of encapsulated agents with a reduced number of production steps. BACKGROUND

[0002] Liposomes are defined as artificial microscopic vesicles consisting of an aqueous core surrounded by one or more concentric phospholipid layers (lamellae) [1]. Liposomes have gained considerable attention as carriers for a wide range of therapeutic agents because they are both non-toxic and biodegradable, as they are composed of naturally occurring substances [2]. Liposomes have broad potential applications, as they are capable of incorporating hydrophilic (in the aqueous compartment), hydrophobic (within the lipid membrane), and amphiphilic (interface of the lipid and aqueous phases) substances [3]. Furthermore, the biologically active materials encapsulated in liposomes are protected from immediate dilution or degradation. For all these reasons, liposomes are the most popular nanotransport systems used since their discovery.

[0003] The widespread use of liposomes for various purposes has created the need to develop effective and reproducible preparation methods with the greatest possible simplicity. There are different methods for the preparation of liposomes, with numerous variations. Due to its simplicity, most laboratories use the thin lipid film hydration method, first described in 1965 [4]. However, the film method tends to be unsuitable for large-scale production. Additionally, there are concerns about the use of chlorinated solvents.

[0004] The ethanol injection method is an interesting technique for scaling up GMP liposome production. It offers several advantages, e.g. simplicity, GMP-compatible solvent, rapid implementation and reproducibility, as well as the fact that it does not cause lipid degradation or oxidative changes. The ethanol injection method was first reported in 1973 by Batzri and Korn [5], as Petition 870210101890, dated 04 / 11 / 2021, page 6 / 165 2 / 63 one of the first alternatives for the preparation of small unilamellar vesicles (SUVs) without sonication. By immediately diluting ethanol in the aqueous phase, lipid molecules precipitate and form planar fragments of bilayers. Through energy dissipation in the system (by agitation and / or ultrasonication), the fragments of these lipid bilayers tend to decrease the exposure of the hydrophobic parts of their molecules to the aqueous environment, resulting in the curvature of these fragments which take on an almost spherical structure. In the following years, several studies investigated the preparation parameters of the ethanol injection technique (lipid concentration and composition, injection speed, temperature of both phases, agitation speed, etc.) on the characteristics of the resulting liposomes (size distribution, zeta potential, drug encapsulation efficiency, etc.) [6].

[0005] In the classic ethanol injection method, the ethanol phase is present in a smaller percentage compared to the aqueous phase, typically 5-10%. After ethanol evaporation, the liposomal dispersion is extruded to reduce vesicle size. In summary, the classic ethanol injection method comprises 3 key steps: 1. Injection of lipids (ethanol) in aqueous phase; 2. Extrusion to reduce the size of liposomes; 3. Removal of non-encapsulated agents.

[0006] In general, the loading of liposomal therapeutic or imaging agents is achieved by passive or active methods: Passive loading involves dissolving dry lipid films in aqueous solutions containing the agent of interest. This approach can only be used for water-soluble agents, and loading efficiency is often low (less than 5%). This method can be used for a wide range of compounds regardless of their chemical structure. Active loading involves the internalization of agents driven by a liposomal transmembrane pH gradient [7]. This process can be extremely effective (greater than 95%), resulting in concentrations Petition 870210101890, dated 04 / 11 / 2021, page 7 / 165 3 / 63 elevated intraliposomal levels and minimal waste of precious chemotherapeutic agents.

[0007] This method (active loading) requires, however, that the molecules have a different protonation state at the extreme pHs of the buffers used inside and outside the liposomes. In this way, a given molecule will diffuse the lipid bilayer when two different pHs are established inside and outside the liposome. Thus, a pH gradient is the driving force for translocating and retaining weak amphiphilic acids and bases [8].

[0008] Also reported in the literature is the approach of active loading for therapeutic or imaging agents of weakly basic amines, using a transmembrane gradient of ammonium sulfate. In this case, the ammonia gradient drives a pH gradient, leading to the active transport of the agent into the liposome. The sulfate then acts as a counter-ion for the ionized agent, causing it to precipitate inside the liposome. This strategy was applied to the production of liposomal doxorubicin in the case of Doxil. Myocet is another example of liposomal doxorubicin that is remotely loaded, although the pH gradient is established with citric acid [9].

[0009] In the active loading process, after liposome preparation using the classic ethanol injection method, the extraliposomal phase is removed, and then the agent is added to the extraliposomal phase and the liposomes are incubated to allow the remote loading process to proceed. In summary, the active loading process comprises 5 key steps: 1. Injection of lipids (ethanol) in aqueous phase; 2. Extrusion to reduce the size of liposomes; 3. Removal of the extraliposomal phase; 4. Incubation of liposomes with agents; 5. Removal of non-encapsulated agents.

[0010] Document WO / 2013 / 084208 (Paulo A. et al., 2013, “Liposomes and its production method”) describes a liposomal production method which is the lipid film hydration method. Petition 870210101890, dated 04 / 11 / 2021, page 8 / 165 4 / 63

[0011] Document US4752425A (Martin F. et al., 1988, “High-encapsulation liposome processing method”) describes a method for producing liposomes using chloroform. The liposomes produced are 1.5 microns or larger, requiring extrusion to reduce their size (where the originally encapsulated agent is lost).

[0012] Document US5549910A (Szoka F. et al., 1994, “Preparation of liposome and lipid complex compositions”) describes a method for obtaining liposomes containing compounds that exhibit low solubility in water, alcohols, and halogenated hydrocarbon solvents. In this method, lipids are dissolved in an aprotic solvent solution, which may additionally contain a short-chain alkanol if solubilization is required. This method requires extrusion to obtain liposomes of a defined size.

[0013] Document US20120171280A1 (Zhang Y, 2011, “Method of making liposomes, liposome compositions made by the methods, and methods of using the same”) describes a method for obtaining liposomes in which the aqueous solution comprises ethylenediaminetetraacetic acid (EDTA) for encapsulation of ascorbic acid or one of its salts. The liposome composition has a selected average particle size diameter of about 200-500 nm.

[0014] US patent 5316771A (Barenholz, Y. et al., 1994, “Method of amphiphatic drug loading in liposomes by ammonium ion gradient”) describes the active loading of weak amphipathic drugs into liposomes using a transmembrane gradient.

[0015] US patent 5939096A (Clerc, SY and Barenholz, 1999, “Stably encapsulating a weak acid drug in liposomes, at a high concentration”) describes liposomes encapsulated with a weak acid drug at a high concentration. The method employed a proton transport mechanism involving the salt of a weak acid to generate a higher inside / lower outside pH gradient.

[0016] Document WO201364911 refers to methods and compositions for producing negatively charged therapeutic polymers encapsulated in lipids, such as nucleic acids, proteins and peptides, which are encapsulated within a lipid layer. Petition 870210101890, dated 04 / 11 / 2021, p. 9 / 165 5 / 63

[0017] Document WO0105374 refers to methods and composition for producing charged therapeutic agent particles encapsulated in a lipid, after mixing pre-formed lipid vesicles, a charged therapeutic agent (with a charge opposite to the lipid) and a destabilizing agent. GENERAL DISCLOSURE OF THE INVENTION

[0018] The method described has the advantage of achieving encapsulation efficiency of small molecules in a targeted liposome equal to or better than previous methods, without extra processing steps to produce nanoparticles. Polycharged molecules, namely those with negative charges in their structure at neutral pHs (5-8), such as methotrexate and doxorubicin, encapsulate better with this method. Methotrexate has high encapsulation rates and doxorubicin with a reduced number of steps. (tables 2 and 3)

[0019] In the proposed alternative method, a higher encapsulation efficacy of the therapeutic or imaging agent is achieved using a preconcentration method with an ethanolic:aqueous phase at a similar volume ratio, and the liposomes can be diluted at the end. The new proposed method has a reduced number of steps (only 2), which is desirable in an industrial process. These characteristics are combined for the first time in the same method, differentiating it from those reported in the literature.

[0020] The widespread use of liposomes for various purposes has created the need to develop preparative methods that must be effective, reproducible, and as simple as possible. Existing methods remain laborious for scaling up industrial production and / or achieve low encapsulation efficiency of the agent of interest. Therefore, it is imperative to develop a method with a reduced number of steps that achieves high encapsulation efficiency of the encapsulated agent.

[0021] The lipids used to produce liposomes can be changed or modified to customize the properties of the liposomal surface and membrane layer. There are different classes of lipids, based on their charge: neutral, cationic, anionic. The addition of organic molecules to the phosphate group of the head creates Petition 870210101890, dated 04 / 11 / 2021, page 10 / 165 6 / 63 a variety of phospholipid species, such as phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG), and phosphatidylcholine (PC). All of these lipids can be used in the production of liposomes.

[0022] Unmodified liposomes do not survive long in circulation, as they are removed by macrophages. One of the first attempts to overcome these problems focused on manipulating lipid membrane components to modify bilayer fluidity, such as by including a steroid. In this way, our liposomal formulations can preferentially contain cholesterol (CH), which can vary from a molar ratio of 35-55%, preferably 35-40%. It has been shown that the incorporation of cholesterol into liposomes reduces interaction with blood proteins, causing increased phospholipid packing in the lipid bilayer.

[0023] In addition, in a preferred embodiment, a synthetic polymer, polyethylene glycol (PEG), was incorporated into the liposomes. Liposomes containing PEG showed less binding to blood proteins, reduced RES uptake, and thus prolonged duration of liposomes in the circulatory system. This extended the bloodstream of conventional liposomes for drug delivery; the DSPE-MPEG conjugated phospholipid was incorporated into a lipid film of these new formulations, in a molar ratio that can vary between 4-12%, preferably 5-10%.

[0024] It has also been shown that liposomes with a surface modified with gangliosides have a prolonged circulation time in the bloodstream compared to unmodified ones. These characteristics are potentially useful for applications of gangliosides in immunotherapies. Several glycolipids were tested in RES uptake studies of liposomes after intravenous injection: the glycolipid GM1 (a monosialoganglioside derived from brain tissue) significantly decreased RES uptake when incorporated into the liposome surface, and the formulation remained in the bloodstream for several hours.

[0025] Active targeting exploits the specific modification of the liposomal surface with a targeting ligand, which can lead to its accumulation at the target site or intracellular delivery to target cells. The inclusion of certain ligands in Petition 870210101890, dated 04 / 11 / 2021, page 11 / 165 7 / 63 liposomes allow the release of their contents intracellularly by receptor-mediated endocytosis. Integration of the targeting agent to the membrane surface can be achieved by conjugation to phospholipid or fatty acyl chains or incorporation into the lipid membrane.

[0026] There are different methods for preparing liposomes, with numerous variations. In the ethanol injection method (5% ethanol), a fraction of the aqueous solution with water-soluble substances is passively encapsulated within vesicles. The advantage of this method is its simplicity, but only a very small percentage of water-soluble therapeutic or imaging agents can be encapsulated in this way.

[0027] In remote loading, empty liposomes are generally prepared in a low pH starter salt or buffer. The extraliposomal phase is then removed using dialysis or size exclusion chromatography, or by titrating the pH to slightly basic conditions. Finally, the agent is added to the extraliposomal phase and the liposomes are incubated to allow the remote loading process to proceed [6]. The number of steps involved makes the production process difficult to scale up, constituting a barrier to further development of this standard approach.

[0028] Thus, we focused our efforts on optimizing the encapsulation of the agent within liposomes, with reduced production steps. In fact, only a small amount of the agent used was encapsulated (e.g., 3-5% for the drug methotrexate), with a large amount of agent being wasted, and this can be a problem in a scale-up process. In order to increase the encapsulated agent, numerous conditions were tested in various steps of the production method. Namely, aqueous phase in organic phase containing phospholipids (instead of the opposite), not removing the ethanol, performing the injection at room temperature instead of 70°C, testing different injection speeds and various concentrations of the organic phase (5% up to 95%).

[0029] The present invention consists of a new method for producing liposomes, in which the hydrophobic components of the liposomes are dissolved in ethanol, and are Petition 870210101890, dated 04 / 11 / 2021, page 12 / 165 8 / 63 injected into an aqueous phase at a rate of approximately 2-4 mL / min under vigorous stirring. The initial volume ratio of ethanol:aqueous phase is 1 / 1. After evaporation of the ethanol or tangential flow filtration, the liposomal dispersion should be diluted 1 to 10 times to the desired final concentration.

[0030] In one embodiment, the pre-concentration method comprises 2 key steps: • Injection of lipids (ethanol) in aqueous phase (1:1 v / v), followed by dilution; • Removal of non-encapsulated agents.

[0031] In one embodiment, this method allows for high encapsulation efficiencies (e.g., ~40%) for polysaturating agents, such as methotrexate, with a reduced number of steps (only 2). The initial pre-concentration (using a lower aqueous volume) increases the phospholipid concentration and consequently allows for higher encapsulation efficiency. Additionally, the use of an initial volume ratio of 1:1 ethanol:aqueous phase allows for an equilibrium between two phases with different polarities, increasing the encapsulation of the agents.

[0032] In one embodiment, the disclosure refers to a method for encapsulating an active ingredient in liposomes comprising the following sequential steps: - Prepare an ethanolic phase by mixing hydrophobic phospholipid molecules and a steroid with ethanol; preferably cholesterol; - Prepare an aqueous phase with an active ingredient and a targeting agent in a buffer solution; - obtain liposomes by injecting the ethanolic phase into the aqueous phase, at a temperature of about 50 °C to about 80 °C, where the volume ratio of the ethanolic / aqueous phases is between 1:1 and 3:2; - remove the ethanol by evaporation or tangential flow filtration; - remove any remaining free active ingredient in an appropriate manner, in particular by tangential flow filtration; where the targeting agent is a peptide that is conjugated to a liposomal component or incorporated into the lipid membrane. Petition 870210101890, dated 04 / 11 / 2021, page 13 / 165 9 / 63

[0033] In another embodiment, the disclosure refers to a method, which additionally comprises the step of diluting the liposomal dispersion 1 to 10 times in a more dilute aqueous phase.

[0034] In another embodiment, the disclosure refers to a method in which the ethanolic phase is injected at a rate of approximately 2-4 mL / minute.

[0035] In another embodiment, the disclosure refers to a method in which the injection step is carried out under agitation.

[0036] In another embodiment, the disclosure refers to a method, in which the active ingredient is a drug, in particular an anticancer drug, an antirheumatic drug, a drug against neurodegenerative diseases, an antioxidant drug, an anti-inflammatory drug, an antipyretic drug, an antibiotic drug, an antiviral drug, an analgesic drug or combinations thereof.

[0037] In another embodiment, disclosure refers to a method wherein the targeting agent is a peptide selected from the following list with a degree of identity of at least 90% to the following sequences: SEQ-ID. No. 1, SEQ-ID. No. 2, SEQ-ID. No. 3, or mixtures thereof; comprising at least one sequence 95%, or preferably at least 96% identical, or at least 97% identical, or at least 98% identical, or at least 99% identical to SEQ-ID. No. 1, SEQ-ID. No. 2, SEQ-ID. No. 3, or mixtures thereof.

[0038] Methods for sequence alignment for comparison are well known in the art, including methods such as GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the Needleman and Wunsch algorithm ((1970) J Mol Biol 48: 443-453) to find the global alignment (along the entire sequence) of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates the percentage of sequence identity and performs a statistical analysis of the similarity between the two sequences. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). Global percentages of similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., Petition 870210101890, dated 04 / 11 / 2021, p. 14 / 165 10 / 63 BMC Bioinformatics. 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences). A small amount of manual editing can be performed to optimize the alignment between conserved motifs, as would be evident to a specialist in the technique. The sequence identity values, which are indicated in this subject matter as a percentage, were determined throughout the amino acid sequence using BLAST with standard parameters.

[0039] In another embodiment, the disclosure refers to a method in which the concentration of ethanol, with respect to the initial aqueous volume, is between 40% and 60%, preferably 50%.

[0040] In another embodiment, the disclosure refers to a method, in which the temperature is 60 °C or 70 °C.

[0041] In another embodiment, the disclosure refers to a method in which the active ingredient is a polycharged molecule containing at least one negative charge at a pH of about 4 to about 7, particularly methotrexate or doxorubicin.

[0042] In another embodiment, the disclosure refers to a method in which the aqueous phase is phosphate-buffered saline solution, PBS.

[0043] In another embodiment, the disclosure refers to a method in which the ethanolic phase comprises anionic, neutral or cationic phospholipids.

[0044] In another embodiment, the disclosure refers to a method, wherein the ethanolic phase comprises phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols and / or their derivatives or mixtures thereof, in particular 1,2-dioleoyl-sunglycerol-3-phosphoethanolamine.

[0045] In another embodiment, disclosure refers to a method in which the ethanolic phase comprises a steroid, a stealth agent, a targeting agent, or a mixture thereof.

[0046] In another embodiment, the disclosure refers to a method in which the steroid is cholesterol, and / or a derivative thereof, in particular cholesteryl hemisuccinate. Petition 870210101890, dated 04 / 11 / 2021, p. 15 / 165 11 / 63

[0047] In another embodiment, disclosure refers to a method in which the stealth agent is polyethylene glycol, PEG, or gangliosides.

[0048] In another embodiment, the disclosure refers to a method in which polyethylene glycol, PEG, is linked to a phospholipid, in particular distearolphosphatidylethanolamine.

[0049] In another embodiment, disclosure refers to a method in which the targeting agent is incorporated into the lipid membrane.

[0050] In another modality, the disclosure refers to a method in which the active ingredient is an imaging or therapeutic agent.

[0051] In another modality, disclosure refers to a method in which the imaging or therapeutic agent is hydrophobic or hydrophilic.

[0052] In another modality, the disclosure refers to a method, in which the imaging agent is a pigment. DETAILED DESCRIPTION

[0053] This disclosure relates to a method for producing liposomes in order to obtain high encapsulation efficiency of the encapsulated agents with a reduced number of production steps, notably avoiding the extrusion step of the classic liposomal production process. The liposomes of the invention are intended to carry a therapeutic agent such as an anticancer, antioxidant, anti-inflammatory, antipyretic, antibiotic, antiviral, antirheumatic, analgesic, growth factor, or mixtures thereof.

[0054] The method described has the advantage of achieving encapsulation efficiency of small molecules in a targeted liposome equal to or better than previous methods without extra processing steps to produce nanoparticles.

[0055] In order to increase the encapsulated agent, with reduced production steps, numerous conditions were tested in various steps of the production method. The results demonstrate that the initial percentage of ethanol significantly affected the encapsulation efficiency. A drastic increase in encapsulation efficiency was noted as the volume of ethanol exceeded the classic 5% (50%). Petition 870210101890, dated 04 / 11 / 2021, page 16 / 165 12 / 63 with respect to the initial aqueous phase), and also using a lower volume of aqueous solution of the agent (the sample is subsequently diluted five times to obtain the usual agent concentration after ethanol evaporation or tangential flow filtration). The size of the vesicles was positively affected by the volume of ethanol after this ratio was achieved. In effect, batches with a higher volume of ethanol (>50%) showed larger vesicles (< 150 nm) than the previously produced liposome batches. These results can be attributed to the slower diffusion of ethanol related to its volume increase in the aqueous phase, leading to the formation of larger liposomes due to the slow self-assembly of phospholipids. Consequently, the smaller the vesicle size, the smaller the volume of the aqueous core and the lower the encapsulation efficiencies obtained, knowing that the hydrophilic agent is mainly encapsulated in the aqueous core of the liposome [10, 11].However, a compromise between encapsulation efficiency and size was achieved using 50% of the initial ethanol volume. Liposomes obtained with this percentage of ethanol exhibit a reduced size (<150 nm) and PDI values ​​(<0.1). Furthermore, under these conditions, the extrusion process typically needed to be reduced, and uniform vesicle size is entirely dispensable. Liposome production

[0056] In one embodiment, liposomes composed of DOPE / Cholesterol / DSP-MPEG (54:36:10 molar ratio) were prepared using the ethanol injection method. Briefly, the lipids (DOPE, cholesterol, and DSPE-MPEG) were dissolved in ethanol (5% in the classic ethanol injection method; 50% in the new proposed pre-concentration method, relative to the initial aqueous phase at 20%) at 60 °C.

[0057] The solution was injected under agitation into an aqueous solution (phosphate-buffered saline, PBS). This process is carried out at 70 °C, continuing for the time necessary to evaporate all the ethanol.

[0058] In the classic ethanol injection method, liposomes are extruded to reduce their size. In the pre-concentration method, after ethanol evaporation or tangential flow filtration, the liposomal dispersion is diluted five times in PBS (the Petition 870210101890, dated 04 / 11 / 2021, page 17 / 165 13 / 63 (the remaining 80% of the volume is added). The free therapeutic or imaging agent that was not incorporated into liposomes was removed from the samples after passing through a gel filtration chromatography column (GE Healthcare) with a 5 kDa cutoff (PD-10 Desalination Columns containing 8.3 mL of Sephadex G-25 Medium). Hydrophilic therapeutic or imaging agents (e.g., methotrexate and doxorubicin) are present in this aqueous phase in the classic ethanol injection method and in the new proposed pre-concentration method. In remote / active loading, after production in ammonium sulfate (120 mM, pH=8.5), the buffer is changed to Trizma®Base sucrose (10% w / v, buffered at pH 9.0) and the empty liposomes are incubated with the therapeutic or imaging agents.

[0059] Characterization of liposomes encapsulating methotrexate: a comparative study between various production methods. Average Z (d.nm) PDI Encapsulation Efficiency (%) Number of Steps Preconcentration Method (50% ethanol relative to initial aqueous buffer) 103.1 ± 2.333 0.117 ± 0.003 43.4 2 (no extrusion) 66% ethanol 343.3 ± 9.136 0.067 ± 0.029 41.6 2 (no extrusion) 66% ethanol 124.7 ± 0.635 0.061 ± 0.009 8.3 3 (extrusion) 33% ethanol 99.66 ± 0.095 0.240 ± 0.006 7.6 3 (extrusion) Classical Injection Method 117.7 ± 1.779 0.053 ± 0.018 5.7 3 (extrusion) Petition 870210101890, dated 04 / 11 / 2021, page 18 / 165 14 / 63 ethanolic (5% ethanol)

[0060] Table II - Characterization of liposomes encapsulating doxorubicin: comparative study between various production methods. Average Z (d.nm) PDI Encapsulation Efficiency (%) Number of Steps Preconcentration Method (50% ethanol relative to initial 20% aqueous buffer) 131.2 ± 1.124 0.109 ± 0.010 84.9 2 (without extrusion) Classical Ethanol Injection Method (5% ethanol) 115.1 ± 0.551 0.048 ± 0.027 85.1 3 (extrusion) Remote / Active Loading 124.8 ± 1.825 0.132 ± 0.020 76.1 5 (extrusion)

[0061] Table III - List from A to Z of drugs for cancer including combination treatments (note: alphabetical order based on English terminology) AL Abemaciclib Lanreotide Acetate Abiraterone Acetate Lapatinib Ditosylate Abraxane (Paclitaxel-Albumin Stabilized Nanoparticle Formulation) Lartruvo (Olaratumab) ABVD Lenalidomide ABVE Lenvatinib Mesylate ABVE-PC Lenvima (Lelvatinib Mesylate) Petition 870210101890, dated 04 / 11 / 2021, page 19 / 165 15 / 63 AC Letrozole Acalabrutinib Leucovorin Calcium AC-T Leukeran (Chlorambucil) Actemra (Tocilizumab) Leuprolide Acetate Adcetris (Brentuximab Vedotin) Levulan Kerastik (Aminolevulinic Acid) ADE Libtayo (Cemiplimab-rwlc) Ado-Trastuzumab Emtansine LipoDox (Liposomes with Doxorubicin Hydrochloride) Adriamycin (Doxorubicin Hydrochloride) Lomustine Afatinib Dimaleate Lonsurf (Trifluridine and Tipiracil Hydrochloride) Afinitor (Everolimus) Lupron (Leuprolide Acetate) Akynzeo (Netupitant and Palonosetron Hydrochloride) Lupron Depot (Acetate of Leuprolide) Aldara (Imiquimod) Lutatera (Lutetium Lu 177 Dotate) Aldesleukin Lutetium (Lu 177 Dotate) Alecensa (Alectinib) Lynparza (Olaparib) Alectinib Alemtuzumab M Alimta (Pemetrexed Disodium) Marqibo (Vincristine Sulfate Liposomes) Aliqopa (Copanlisib Hydrochloride) Matulane (Procarbazine Hydrochloride) Alkeran Injectable (Melphalan Hydrochloride) Mechlorethamine Hydrochloride Alkeran Tablets(Melfalano) Megestrol Acetate Aloxi (Palonosetron Hydrochloride) Mekinist (Trametinibe) Alunbrig (Brigatinibe) Mektovi (Binimetinibe) Petition 870210101890, of 04 / 11 / 2021, p. 20 / 165 16 / 63 Ameluz (Aminolevulinic Acid) Melphalan Amifostine Melphalan Hydrochloride Aminolevulinic Acid Mercaptopurine Anastrozole Mesna Apalutamide Mesnex (Mesna) Aprepitant Methotrexate Aranesp (Darbepoetin Alfa) Methylnaltrexone Bromide Aredia (Pamidronate Disodium) Midostaurin Arimidex (Anastrozole) Mitomycin C Aromasin (Exemestane) Mitoxantrone Hydrochloride Arranon (Nelarabine) Mogamulizumab-kpkc Arsenic Trioxide Mozobil (Plerixafor) Arzerra (Ofatumumab) Mustargen (Mechlorethamine Hydrochloride) Erwinia chrysanthemi Asparaginase MVAC Atezolizumab Myleran (Busulfan) Avastin (Bevacizumab) Mylotarg (Gentuzumab Ozogamicin) Avelumab Axicabtagen Ciloleucel N Axitinib Paclitaxel Nanoparticles (Paclitaxel-Albumin Stabilized Nanoparticle Formulation) Azacitidine Navelbine (Vinorelbine Tartrate) Azedra (Iobenguane I 131) Necitumumab Nelarabine B Neratinib Maleate Bavencio (Avelumab) Nerlynx (Neratinib Maleate) Petition 870210101890, of 04 / 11 / 2021, p. 21 / 165 17 / 63 BEACOPP Netupitanto e Chloridrato de Palonosetrona Beleodaq (Belinostat) Neulasta (Pegfilgrastim) Belinostat Neupogen (Filgrastim) Chloridrato de Bendamustina Nexavar (Tosilato de Sorafenib) Bendeka (Tosilato de Bendamustina) Nilandron (Nilutamida) BEP Nilotinibe Besponsa (Inotuzumabe Ozogamicina) Nilutamida Bevacizumabe Ninlaro (Citrato de Ixazomibe) Bexaroteno Tosilato de Niraparibe Monoidratado Bicalutamida Nivolumabe BiCNU (Carmustina) Nplate (Romiplostim) Binimetinibe Bleomicina O Blinatumomabe Obinutuzumabe Blincyto (Blinatumomabe) Odomzo (Sonidegibe) Bortezomibe OEPA Bosulif (Bosutinabe) Ofatumumab Bosutinabe OFF Braftovi (Encorafenibe) Olaparibe Brentuximabe Vedotin Olaratumabe Brigatinibe Mepessuccinato de Omacetaxa BuMel Oncaspar (Pegaspargase) Bussulfano Cloridrato de Ondansetrona Busulfex (Bussulfano) Onivyde (Lipossomas com Cloridrato de Irinotecano) Ontak (Denileucina Diftitox) Petition 870210101890, dated 04 / 11 / 2021, page 22 / 165 18 / 63 C Opdivo (Nivolumab) Cabazitaxel OPPA Cabometyx (Cabozantinib S-Malate) Osimertinib Cabozantinib S-Malate Oxaliplatin CAF Calquence (Acalabrutinib) P Campath (Alemtuzumab) Paclitaxel Camptosar (Irinotecan Hydrochloride) Paclitaxel-Albumin Stabilized Nanoparticle Formulation Capecitabine PAD CAPOX Palbociclib Carac (Fluorouracil - Topical) Palifermine Carboplatin Palonosetron Hydrochloride CARBOPLATIN-TAXOL Palonosetron Hydrochloride and Netupitant Carfilzomib Pamidronate Disodium Carmustine Panitumumab Carmustine in Implant Form Panobinostat Casodex (Bicalutamide) Pazopanib Hydrochloride CEM PCV Cemiplimab-rwlc PEB Ceritinib Pegaspargase Cerubidine (Daunorubicin Hydrochloride) Pegfilgrastim Cervarix (Bivalent Recombinant HPV Vaccine) Peginterferon Alfa-2b Cetuximab PEG-Intron (Peginterferon Alfa-2b) CEV Pembrolizumab Petition 870210101890, dated 04 / 11 / 2021, p. 23 / 165 19 / 63 Chlorambucil Pemetrexed Disodium Chlorambucil-Prednisone Perjeta (Pertuzumab) CHOP Pertuzumab Cisplatin Plerixafor Cladribine Pomalidomide Clofarabine Pomalyst (Pomalidomide) Clolar (Clofarabine) Ponatinib Hydrochloride CMF Portrazza (Necitumumab) Cobimetinib Poteligeo (Mogamulizumab-kpkc) Cometriq (Cabozantinib S-Malate) Pralatrexate Copanlisib Hydrochloride Prednisone COPDAC Procarbazine Hydrochloride Copiktra (Duvelisib) Procrit (Epoetin Alfa) COPP Proleukin (Aldesleukin) COPP-ABV Prolia (Denosumab) Cosmegen (Dactinomycin) Promacta (Eltrombopag Olamine) Cotellic (Cobimetinib) Propranolol Hydrochloride Crizotinib Provenge (Sipuleucel-T) CVP Purinetol (Mercaptopurine) Cyclophosphamide Purixan (Mercaptopurine) Cyramza (Ramucirumab) Cytarabine Q Cytarabine Liposomes [No Entries] Cytosar-U (Cytarabine) RD Radium-223 Dichloride Dabrafenib Raloxifene Hydrochloride Dacarbazine Ramucirumab Petition 870210101890, dated 04 / 11 / 2021, page 24 / 165 20 / 63 Dacogen (Decitabine) Rasburicase Dacomitinib R-CHOP Dactinomycin R-CVP Daratumumab Recombinant Bivalent Human Papillomavirus (HPV) Vaccine Darbepoetin Alfa Recombinant Nonavalent Human Papillomavirus (HPV) Vaccine Darzalex (Daratumumab) Recombinant Quadrivalent Human Papillomavirus (HPV) Vaccine Dasatinib Recombinant Interferon Alfa-2b Daunorubicin Hydrochloride Regorafenib Liposomes with Daunorubicin Hydrochloride and Cytarabine Relistor (Methylnaltrexone Bromide) Decitabine R-EPOCH Defibrotide Sodium Retacrit (Epoetin Alfa) Defitelio (Defibrotide Sodium) Revlimid (Lenalidomide) Degarelix Rheumatrex (Methotrexate) Denileukin Diftitox Ribociclib Denosumab R-ICE DepoCyt (Cytarabine Liposomes) Rituxan (Rituximab) Dexamethasone Rituxan Hycela (Rituximab and Human Hyaluronidase) Dexrazoxane Hydrochloride Rituximab Dinutuximab Rituximab and Human Hyaluronidase Docetaxel Rolapitant Hydrochloride Petition 870210101890, dated 04 / 11 / 2021, p. 25 / 165 21 / 63 Doxil (Liposomes with Doxorubicin Hydrochloride) Romidepsin Doxorubicin Hydrochloride Romiplostim Liposomes with Doxorubicin Hydrochloride Rubidomycin (Daunorubicin Hydrochloride) Dox-SL (Liposomes with Doxorubicin Hydrochloride) Rubraca (Rucaparib Camsylate) Durvalumab Rucaparib Camsylate Duvelisib Ruxolitinib Phosphate Rydapt (Midostaurin) Efudex (Fluorouracil - Topical) Eligard (Leuprolide Acetate) Sancuso (Granisetron) Elitek (Rasburicase) Sclerosol Intrapleural Aerosol (Talc) Ellence (Epirubicin Hydrochloride) Siltuximab Elotuzumab Sipuleucel-T Eloxatin (Oxaliplatin) Somatuline Depot (Lanreotide Acetate) Eltrombopag Olamine Sonidegib Emend (Aprepitant) Sorafenib Tosylate Empliciti (Elotuzumab) Sprycel (Dasatinib) Enasidenib Mesylate STANFORD V Encorafenib Sterile Talcum Powder (Talc) Enzalutamide Steritalc (Talc) Epirubicin Hydrochloride Stivarga (Regorafenib) EPOCH Sunitinib Malate Epoetin Alfa Sustol(Granisetron) Epogen (Epoetin Alfa) Sutent (Sunitinib Malate) Petition 870210101890, dated 04 / 11 / 2021, page 26 / 165 22 / 63 Erbitux (Cetuximabe) Sylatron (Peginterferon Alfa-2b) Mesylato de Eribulina Sylvant (Siltuximabe) Erivedge (Vismodegib) Synribo (Mepessuccinato de Omacetaxina) Erleada (Apalutamida) Hydrochloride de Erlotinibe T Erwinaze (Asparaginase de Erwinia chrysanthemi) Tabloid (Thioguanine) Etiol (Amifostine) TAC Etopophos (Fosfato de Etoposídeo) Tafinlar (Dabrafenibe) Etoposídeo Tagrisso (Osimertinibe) Fosfato de Etoposídeo Talco Evacet (Lipossomas com Cloridrato de Doxorrubicina) Talimogene Laherparepvec Everolímus Citrato de Tamoxifeno Evista (Hydrochloride de Raloxifeno) Tarabine PFS (Cytarabine) Evomela (Melphalan Hydrochloride) Tarceva (Erlotinibe Hydrochloride) Exemestane Targretin (Bexaroteno) Tasigna (Nilotinibe) F Tavalisse (Fostamatinibe Dissódico) 5-FU (Fluorouracil Injetável) Taxol (Paclitaxel) 5-FU (Fluorouracil--Tópico) Taxotere (Docetaxel) Fareston (Toremifeno) Tecentriq (Atezolizumabe) Farydak (Panobinostat) Temodar (Temozolomida) Faslodex (Fulvestranto) Temozolomida FEC TemsirolímusFemara (Letrozole) Thalidomide Filgrastim Thalomid (Thalidomide) Firmagon (Degarelix) Thioguanine Petition 870210101890, dated 04 / 11 / 2021, page 27 / 165 23 / 63 Fludarabine Phosphate Thiotepa Fluoroplex (Fluorouracil - Topical) Tibsovo (Ivosidenib) Fluorouracil Injection Tisagenlecleucel Fluorouracil - Topical Tocilizumab Flutamide Tolak (Fluorouracil - Topical) FOLFIRI Topotecan Hydrochloride FOLFIRI-BEVACIZUMAB Toremifene FOLFIRI-CETUXIMAB Torisel (Temsirolimus) FOLFIRINOX Totect (Dexrazoxane Hydrochloride) FOLFOX TPF Folotyn (Pralatrexate) Trabectedin Fostamatinib Disodium Trametinib FU-LV Trastuzumab Fulvestrant Treanda (Bendamustine Hydrochloride) Fusilev (Leucovorin Calcium) Trexall (Methotrexate) Trifluridine and Tipiracil Hydrochloride G Trisenox (Arsenic Trioxide) Gardasil (Recombinant Quadrivalent HPV Vaccine) Tykerb (Lapatinib Ditosylate) Gardasil 9 (Recombinant Nonavalent HPV Vaccine) Gazyva (Obinutuzumab) U Gefitinib Unituxin (Dinutuximab) Gemcitabine Hydrochloride Uridine Triacetate GENCITABINE-CISPLATIN GENCITABINE-OXALIPLATIN V Gemtuzumab Ozogamicin VAC Gemzar (Gemcitabine Hydrochloride)Valrubicin Petition 870210101890, dated 04 / 11 / 2021, page 28 / 165 24 / 63 Gilotrif (Afatinib Dimaleate) Valstar (Valrubicin) Gleevec (Imatinib Mesylate) Vandetanib Gliadel Wafer (Carmustine Implant) VAMP Glucarpidase Varubi (Rolapitant Hydrochloride) Goserelin Acetate Vectibix (Panitumumab) Granisetron VeIP Granisetron Hydrochloride Velcade (Bortezomib) Granix (Filgrastim) Vemurafenib Venclexta (Venetoclax) H Venetoclax Halaven (Eribulin Mesylate) Verzenio (Abemaciclib) Hemangeol (Propranolol Hydrochloride) Vidaza (Azacytidine) Herceptin (Trastuzumab) Vinblastine Sulfate Bivalent HPV Vaccine, Recombinant Vincristine Nonavalent HPV Vaccine, Recombinant Liposomes with Vincristine Sulfate Quadrivalent HPV VaccineRecombinant Vinorelbine Tartarate Hycamtin (Topotecan Hydrochloride) VIP Hydrea (Hydroxiureia) Vismodegibe Hidroxiureia Vistogard (Uridine Triacetate) Hyper-CVAD Vizimpro (Dacomitinibe) Voraxaze (Glucarpidase) I Vorinostat Ibrance (Palbociclibe) Votrient (Pazopanibe Hydrochloride), Petition 870210101890, 04 / 11 / 2021, pág. 29 / 165 25 / 63 Ibrytumomab Thiouxetane Vyxeos (Liposomes with Daunorubicin and Cytarabine Hydrochloride) Ibrutinibe ICE W Iclusig (Ponatinibe Hydrochloride) [No Entries] Hydarrubicin Idelalisibe X Ifdhifa Hydrochloride (Mesylase X Idhifa (Mesylate X) enanacosideribene (Ifosfamide) Xeloda (Capecitabine) Ifosfamide XELIRI IL-2 (Aldesleucine) XELOX Imatinibe Xgeva Mesylate (Denosumabe) Imbruvica (Ibrutinibe) Xofigo (Radium Dichloride 223) Imphinzi (DurvalugiEEquinzademidabe) Xtamida Laherparepvec) Y Inlyta (Axitinibe) Yervoy (Ipilimumabe) Inotuzumabe Ozogamicina Yescarta (Axicabtagene Ciloleucel) Interferon Alpha-2b,Recombinant Yondelis (Trabectedin) Interleukin-2 (Aldesleukin) Intron A (Recombinant Interferon Alpha-2b) Z IobenguaneI 131 Zaltrap (Ziv-Aflibercept) Ipilimumab Zarxio (Filgrastim) Iressa (Gefitinib) Zejula (Niraparib Tosylate Monohydrate) Irinotecan Hydrochloride Zelboraf (Vemurafenib) Irinotecan Hydrochloride Liposomes Zevalin (Ibritumomab Thiuxeta) Petition 870210101890, dated 04 / 11 / 2021, page 30 / 165 26 / 63 Istodax (Romidepsin) Zinecard (Dexrazoxane Hydrochloride) Ivosidenib Ziv-Aflibercept Ixabepilone Zofran (Ondansetron Hydrochloride) Ixazomib Citrate Zoladex (Goserelin Acetate) Ixempra (Ixabepilone) Zoledronic Acid Zolinza (Vorinostat) J Zometa (Zoledronic Acid) Jakafi (Ruxolitinib Phosphate) Zydelig (Idelalisib) JEB Zykadia (Ceritinib) Jevtana (Cabazitaxel) Zytiga (Abiraterone Acetate) K Kadcyla (Ado-Trastuzumab Emtansine) Kepivance (Palifermin) Keytruda (Pembrolizumab) Kisqali (Ribociclib) Kymriah (Tisagenlecleucel) Kyprolis (Carfilzomib)

[0062] Table IV - List from A to Z of drugs used in therapy for rheumatoid arthritis (note: alphabetical order based on English terminology) AM Abaloparatide (parathyroid hormone) Magnacet Abatacept Maxidone Acetaminophen (children and infants) Meclofenamate sodium Acetaminophen 325mg Mediproxen Acetaminophen 500 mg Medrol Acetaminophen 650 mg Mefenamic acid Acetaminophen with codeine Meloxicam Petition 870210101890, dated 04 / 11 / 2021, page 31 / 165 27 / 63 Acetylsalicylic acid (aspirin) Menest Actemra Menostar Activella Methadone hydrochloride Actonel Metadose Adalimumab Methotrexate Addaprin Methylprednisolone Advil Miacalcin Alendronate Millipred Alendronate with vitamin D Milnacipran Aleve Mitigare Allopurinol Mobic Ambien Morphine sulfate Ambien CR Morphine sulfate oral solution Amitriptyline hydrochloride Morphine sulfate with naltrexone Amrix Motrin Anacin Motrin IB Anacin (aspirin-free) MS Contin Anakinra Mycophenolate mofetil Anaprox Anaprox DS N Apremilast Nabumetone Arava Nalfon Arthrotec Naprelano Atelvia Naprosyn Avinza Naproxen and esomeprazole magnesium Azasan Naproxen sodium (over-the-counter) Azathioprine Naproxen sodium (prescription) Petition 870210101890, dated 04 / 11 / 2021, page 32 / 165 28 / 63 Azulfidine Neoral Azulfidine EN-Pills Neurontin Norco B Baricitinib O Baycadron Olumiant Bayer Opana Belimumab Oramorph SR Benlysta Orapred Betamethasone Orencia Binosto Otezla Boniva Otrexup Brisdelle Oxaprozin Bufferin Oxycodone Oxycodone hydrochloride with acetaminophen C Oxycodone with aspirin Calcitonin (nasal spray) Oxycodone with ibuprofen Cambia OxyContin Canakinumab Oxymorphone hydrochloride Cataflam Celebrex P Celecoxib Paroxetine Celestone Paxil CellCept Fever Reducer / Analgesic PediaCare Cequa (solution) Pediapred Certolizumab pegol Pegloticase Cevimeline Pennsaid Petition 870210101890, dated 04 / 11 / 2021, page 33 / 165 29 / 63 Tylenol for children Percocet Cimzia Percodan Climara Pro Pexeva Clinoril Pilocarpine Cocet Piroxicam Cocet Plus Plaquenil Colchicine Ponstel Colcrys Prednisolone Combunox Prednisone Conjugated estrogens / bazedoxifene Prednisone Intensol ConZip Pregabalin Cortef Prelona Cortisone acetate Premphase Cosentyx Prempro Cyclobenzaprine Primlev Cyclophosphamide Probenecid Cyclosporine Probenecid and colchicine Cyclosporine ophthalmic emulsion / solution Prolia Cymbalta Prozac DR Daypro Raloxifene hydrochloride Denosumab Rasuvo Dexamethasone Rayos DexPak Reclast Diclofenac Remicade Diclofenac potassium Renflexis (infliximab-abda, biosimilar to Remicade) Petition 870210101890, dated 04 / 11 / 2021, p. 34 / 165 30 / 63 Diclofenac sodium Reprexain Diclofenac Sodium liquid / gel Restasis (emulsion) Diclofenac sodium with misoprostol Rheumatrex Diflunisal Risedronate sodium Dolophine Rituxan Duavee Rituximab Duexis Roxicet Duloxetine Roxicodone Dyspel Rybix ODT Ryzolt E EC-Naprosyn S Ecotrin Salagen Effexor XR Sandimmun Embeda Sarafem Enbrel Sarilumab Endocet Savella Endodan Secukinumab Estrace Sertraline Estratabe Simponi, Simponi Aria Estrogens Stelara Estrogens with progesterone Sulfasalazine Etanercept Sulfazine Etodolac Sulfazine EC Evista Sulindac Evoxac Excedrin T Taltz Petition 870210101890, dated 04 / 11 / 2021, page 35 / 165 31 / 63 F Teriparatide (parathyroid hormone) Febuxostat TH Ibuprofen Feldene Terafeldamine Fenoprofen calcium Tivorbex FeverAll Tocilizumab Fexmid Tofacitinib (extended release) Flexeril Tofacitinib (immediate release) Fluoxetine Tolmetin sodium Flurbiprofen Tramadol Forteo Tramadol (extended release) Fortical Tramadol with acetaminophen Fosamax Trexall Fosamax Plus D Tylenol for Arthritic Pain Tylenol Extra Strength G Tylenol Regular Strength Gabapentin Tylenol with Codeine No. 2 Gengraf Tylenol with Codeine No. 3 Genpril Tylenol with Codeine No. 4 Golimumab Tymlos Gralise UH Uloric Horizant Ultracet Humira Ultram Hycet Ultram-ER Hydrocet Ustekinumab Hydrocodone bitartrate Petition 870210101890, dated 04 / 11 / 2021, p. 36 / 165 32 / 63 Hydrocodone bitartrate with acetaminophen V Hydrocodone bitartrate with ibuprofen Venlafaxine Hydrocortisone Veripred 20 Hydrogesic Vicodin Hydroxychloroquine sulfate Vicoprofen Hydroxypropylcellulose lozenges Vimovo Hysingla ER Vivlodex Voltaren I Voltaren XR Ibandronate Ibuprofen (over-the-counter) X Ibuprofen (prescription) Xatmep Ibuprofen with famotidine Xeljanz Ilaris Xeljanz XR Imuran Xodol Indocin Xolox Indomethacin Tylenol for babies Z Inflectra (infliximab-dyyb, biosimilar to Remicade) Zamicet Infliximab Zipsor Intermezzo Zohydro ER I-Prin Zoledronic acid ixekizumab Zoloft Zolpidem K Zolvit Kadian Zometa Petition 870210101890, dated 04 / 11 / 2021, page 37 / 165 33 / 63 Ketoprofen Zorvolex Kevzara Zurampic Kineret Zydone Krystexxa Zyloprim KS Ibuprofeno L Lacrisert Leflunomida lesinurade Lorcet Lortab Lyrica

[0063] Table IV - List of pigments from A to Z (note: alphabetical order based on English terminology) AG Analytical Standard Acetaldehyde-2,4-dinitrophenylhydrazone, for environmental analysis 2-(β-D-Galactosidoxy)naphthol AS-LC Disodium salt of 5-acetamido-3[4-[3-[4-(2,4-di-tert-pentylphenoxy)butylcarbamoyl]-4-hydroxy-1-naphthoxy]phenylazo]-4-hydroxy-2,7-naphthalenedisulfonic acid, Pigment content 90% Gallocyanin, Pigment content 90% Environmental Standard Acetone-2,4-dinitrophenylhydrazone, 99% Gentian Violet for microscopy (Bact., Hist.) 3-Acetylnoradamantane technical grade, 85% Gentian Violet meets the technical specifications of USP tests Petition 870210101890, dated 04 / 11 / 2021, page 38 / 165 34 / 63 Acid Blue 25, 45% pigment content. Technical grade Giemsa stain, used as a blood stain. Acid Blue 29, 40% pigment content. Giemsa stain certified by the Biological Stain Commission. Acid Blue 80, 40% pigment content. Giemsa stain, Modified Solution (for staining (of blood cellular components and blood parasites)). Acid Blue 113, 50% pigment content. Analytical standard. Bis(2,4-dinitrophenylhydrazone) glutaraldehyde, for environmental analysis. Acid Blue 129, 25% pigment content. Guinea Green B, 50% pigment content. Acid Fuchsin used in tissue staining. H Calcium salt of Acid Fuchsin certified by the Biological Stain Commission, Pigment content >60%. H+LC:L

[49] Kematein for microscopy (Hist.Acid Green 25, Pigment content >60% Hematoxylin; Acid Orange 8, Pigment content 65% Hematoxylin certified by the Biological Stain Commission; Acid Red 1, Pigment content 60% 1-heptyl-4-(4-pyridyl)pyridinium bromide at 95%; Acid Red 183, Pigment content 30% 6-hexadecanoyl-2-(((2(trimethylammonium)ethyl)methyl)amino)naphthalene chloride solid; Acid Violet 7, Pigment content 40% 4'-hydroxy-4-biphenylcarboxylic acid at 99%. Petition 870210101890, dated 04 / 11 / 2021, page 39 / 165 35 / 63 Acid Yellow 17, Pigment content 60% 1-(2-Hydroxyethyl)-1,2,3,4-tetrahydro-2,2,4,7-tetramethylquinoline 97% Acid Yellow 25, Pigment content 40% 8-Hydroxyjulolidine 97% Acridine 97% Disodium salt of hydroxynaphthol blue, ACS reagent Acridine Orange hemi(zinc chloride) salt, Pigment content 90% 2-Hydroxy-1,4-naphthoquinone 97% Hydrated Acridine Orange hydrochloride >98% (HPLC) 2-(4-Hydroxyphenyl)-5-pyrimidinol 90% Acridine Orange hydrochloride in solution at 10 mg / mL in H2O Internal salt of Hydroxide 1-(4-hydroxyphenyl)-2,4,6-triphenylpyridinium hydrate 97% Acridine Orange and 10nonyl bromide I Acriflavine Immersion oil with a viscosity of 150 cSt (lit.) Acriflavine hydrochloride Immersion oil with a viscosity of 1,250 cSt (lit.) Adrenochrome Synthetic indigo, Pigment content of 95% ADVASEP™-7 solid Indigo carmine for microscopy (Bact., Hist.), indicator (pH 11.5-14.0) Alcian Blue in 1% solution in 3% acetic acid, pH 2.5 Indigo carmine certified by the Biological Stain Commission, Pigment content of 85% Alcian Blue 8GX powder Indophenol Alcian Blue 8GX certified by the Biological Stain Commission Indophenol Blue, Pigment content of 60%. Petition 870210101890, dated 04 / 11 / 2021, page 40 / 165 36 / 63 Alcian Blue 8GX for microscopy (Bact., Bot., Hist.) Indoxyl-D-galactopyranoside Alcian Blue 8GX in solution for microscopy, 1% in solution (in 3% acetic acid) Practical grade Induline B Alcian Blue, Pyridine variant pigment, Content >85% 98% 1-Iodo-3,5-dinitrobenzene Alexa Fluoride 350 Iodonitrotetrazolium chloride, Used in colorimetric assays.Alexa Fluoride 405 5-Iodosalicylaldehyde 97% Alexa Fluoride 488 IR-797 Chloride, 70% Pigment Content Alexa Fluoride 532 Isopentyl Nitrite 96% Alexa Fluoride 546 4-(4-Isothiocyanatophenylazo)-N,N-dimethylaniline 97% Alexa Fluoride 555 J Alexa Fluoride 568 Janus Green B certified by the Biological Stain Commission, 65% Pigment Content Alexa Fluoride 594 JC-1 Solid Alexa Fluoride 647 Jenner's Dye suitable for blood staining Alexa Fluoride 680 Jenner's Dye certified by the Biological Stain Commission Alexa Fluoride 750 K Alizarin, 97% Pigment Content Keratin Azure Blue Alizarin Black BL Alizarin Red S certified by the Biological Stain Commission Lacmoid. Petition 870210101890, dated 04 / 11 / 2021, page 41 / 165 37 / 63 Alizarin Yellow GG, 50% pigment content; Leishman's stain used as a histological stain; Allophycocyanin Milk-Silver for electron microscopy; Allura Red AC, 80% pigment content; Leucoberbelin Blue I, 65% pigment content; Amaranth, 85-95% pigment content; Leucocrystal Violet; p-Amidinophenyl-p-(6-amidin-2-indolyl)phenyl ether dihydrochloride; Leucomalachite Green; 97% 1-Aminoanthraquinone; Light Green SF Yellowish Crystalline; Monosodium salt of 4-Amino-1,1'-azobenzene-3,4'-disulfonic acid, 95% pigment content; Light Green SF Yellowish certified by the Biological Stain Commission; 3-Amino-3-deoxydoxygenin hemisuccinamide, ester Lissamine Green™ B, Pigment content 60% N-(4-Amino-2,5-diethoxyphenyl)benzamide Lithium carbonate pure., ACS reagent, reagent (for microscopy), >99.0% (T) 2-Amino-5,6-dimethylbenzimidazole at 97% White acrylic resin medium LR Dipotassium salt of 4-amino-3,6-disulfo-1,8-naphthalic anhydride Fluorescent dye Dilithium salt of Lucifer Yellow CH N-(5-aminopentyl)biotinamide trifluoroacetate salt solid Dilithium salt of Lucifer Yellow VS at ~85% 2-(3-Aminophenylsulfonyl)ethanol hydrochloride at 97% Lugol's solution for microscopy (Bact., Bot.) 4-Aminophthalonitrile at 98% Lumichrome Trisodium salt of 8-aminopyrene-1,3,6-trisulfonic acid >96.0% (HPCE), solid M. Petition 870210101890, dated 04 / 11 / 2021, page 42 / 165 38 / 63 Diammonium salt of Aniline Blue certified by the Biological Stain Commission; Oxalate salt of Malachite Green, Technical Grade; Aniline Blue in 2.5% solution in 2% acetic acid; Oxalate salt of Malachite Green certified by the Biological Stain Commission; N-[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]aniline monohydrochloride at 94%; Malachite Green Carbinol hydrochloride, 85% pigment content; 8-anilino-1-naphthalenesulfonic acid; Malonaldehyde bis(phenylimine) monohydrochloride at 97%; Hemimagnesium salt of hydrated 8-anilino-1-naphthalenesulfonic acid for fluorescence, >95% (titration with perchloric acid); Martius Yellow, 85% pigment content; Anisaldehyde in solution; Sodium salt of Yellow. Martius monohydrate 98% Anthrone, ACS reagent, 97% May-Grünwald stain Arsenazo III, calcium-sensitive pigment Melanin from Sepia officinalis Astrazon Orange G Metanila Yellow for microscopy (Hist.), indicator (pH 1.2-2.3) Auramine O, Pigment content >80%, certified by the Biological Stain Commission Metanil Yellow, Pigment content of 70% 1-Azidoadamantane at 97% 4-(4-Methoxybenzylamino)-7-nitrobenzofurazane Azobenzene at 98% 4-Methoxydiphenyl at 97% Azocarmine G 3-Methoxydiphenylamine at 98%. Petition 870210101890, dated 04 / 11 / 2021, page 43 / 165 39 / 63 Monosodium salt of Azomethine-H hydrate at ~95% 1-methoxy-5-methylphenazinium methylsulfate >95% Azofloxine for microscopy (Hist.) 2-Methoxy-N4-phenyl-1,4-phenylenediamine at 95% Azure B certified by the Biological Stain Commission 6-Methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4b]indole at 97% Azure B prepared by direct synthesis 6-methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indole hydrochloride at 97% Azure A chloride, Pigment content >70% 6-methoxy-1,2,3,4-tetrahydro-9H-pyrido[3,4-b]indol-1-carboxylic acid at 97% Azure A chloride certified by Biological Stain Commission 2-Methyl-2-adamantanol 97% Azure Eosinate A 1-(Methylamino)anthraquinone 98% Azure Eosinate B 3-Amino-5,6-dichloro-2-pyrazinocarboxylate 97% Azure II powder Methyl Blue Azure Eosinate II Methyl Blue certified by the Biological Stain Commission Azure Tetrafluoroborate B, Pigment content 95% 4,4'-Methylenebis(N,N-dimethylaniline) at 98% B Methylene Blue certified by the Biological Stain Commission Rhodopseudomonas sphaeroides Bacteriochlorophyll Hydrated Methylene Blue suitable for nucleic acid staining, BioReagent Basacryl GL Red, Pigment content of 19% Methylene Blue in solution for microscopy, concentrated according to Ehrlich, concentrated, aqueous solution Basic Blue 3, Pigment content of 25% Methylene Blue in solution at 1.4% (w / v) in 95% ethanol, Petition 870210101890, dated 04 / 11 / 2021, page 44 / 165 40 / 63 Basic Blue 41, 40% pigment content Methylene Blue in solution for microbiology Basic Fuchsin for microscopy (Bact., Bot., Hist.), indicator (pH 1.0-3.1) Fluorogenic reagent 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride Special Basic Fuchsin for flagella, certified Methylene Violet (Bernthsen) certified by the Biological Stain Commission, Pigment content >65% Basic Fuchsin certified by the Biological Stain Commission, Pigment content >88% Methylene Violet 3RAX, 90% pigment content Basic Fuchsin, Pigment content >85% Methyl Green, zinc chloride salt, ~85% Disodium salt of batocuproin disulfonic acid for detection Spectrophotometric analysis of Cu, Fe Methyl Green, zinc chloride salt, for microscopy (Bact., Bot., Hist.)Disodium salt of hydrated batophenanthrolinedisulfonic acid >95% Methyl Green, zinc chloride salt, certified by the Biological Stain Commission, Pigment content 85% Environmental standard Benzaldehyde-2,4-dinitrophenylhydrazone, 99% Methyl Green, zinc chloride salt, <0.5% Crystal Violet, Pigment content 80% Benzaldehyde tosylhydrazone at 98% 4-Methyl-3-nitrobenzyl chloride at 97% Benzidine >98.0% (N) Methyl Orange, ACS reagent, Pigment content 85% Benzidine ISOPAC®, >98.0% (N) 4-Methylphthalonitrile at 99% Benzidine dihydrochloride >99% (titration) Methyl Purple in H2O Benzophenone imine 95% Methyl Red, ACS reagent, crystalline. Petition 870210101890, dated 04 / 11 / 2021, page 45 / 165 41 / 63 99% N-Benzylideneaniline Methyl Red Hydrochloride, ACS reagent 97% N-Benzylidenebenzenesulfonamide Sodium salt of Crystalline Methyl Red N-Benzylidenebenzylamine containing 100 ppm MEHQ as stabilizer, 99% Sodium salt of Methyl Red, ACS reagent, 95% pigment content Biotin-4-Fluorescein Methyl Violet 2B certified by the Biological Stain Commission Bisbenzimide H 33258 >98% (HPLC and TLC) Methyl Violet B base, 85% pigment content Bisbenzimide trihydrochloride H 33342 >98% (HPLC and TLC) Blue Mordent 9, 50% pigment content Bis(cyclohexanone)oxaldihydrazone Orange Mordent 1, Pigment content of 70% Bis(1,3-dibutylbarbituric acid) trimethine oxonol >95% (HPLC) Morin hydrate for microscopy (Fl.), for determination of Al, Be, Zn, Ga, In, Sc, 1-2 mol / mol water N,N'-Bis(2,5-di-tert-butylphenyl)-3,4,9,10-perylenedicarboximide, Pigment content 97% MTT formazan powder 1,3-bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutenediyl bis(internal salt), Pigment content 90% Murexide, ACS reagent [4-[Bis(2-hydroxyethyl)amino]phenyl]-1,1,2-ethylenetricarbonitrile 98% N Bismarck Brown R for microscopy (Bact., Hist.) 2,3-Naphthalenedicarbonitrile 97%. Petition 870210101890, dated 04 / 11 / 2021, p. 46 / 165 42 / 63 Bismarck Brown Y certified by the Biological Stain Commission, Pigment content 50% 2,3-Naphthalenedicarboximide 97% N,N'-Bis(salicylidene)-1,2-phenylenediamine 97% α-Naphtholbenzein, indicator (pH 8.2-10.0) Brilliant Black BN, Pigment content 60% α-Naphtholbenzein, indicator grade Brilliant Blue G in solution, Concentrated Naphthol Black Blue, Pigment content ~50% Brilliant Blue R, Pigment content ~50%, Technical grade Naphthol Black Blue, Pigment content 80% Pure Brilliant Blue R Naphthol Green B, Technical grade Pure Brilliant Blue G Naphthol Green B for microscopy (Hist.), for complexometry Brilliant Blue R Concentrate suitable for SDS-PAGE, methanolic solution Disodium phosphate salt Naphthol AS-GR Brilliant Blue R, Staining solution, ethanolic solution Naphthol phosphate disodium salt AS-MX, phosphatase substrate 'Brilliant Cresyl Blue' for microscopy (Vit.), mixture of toluidine blue and water-blue Naphthol Phosphate AS Brilliant Cresyl Blue ALD Certified by the Biological Stain Commission Disodium salt of naphthol phosphate AS Brilliant Cresyl Blue ALD certified by the Biological Stain Commission α-Naphtholphthalein, practical grade Brilliant Green, Pigment content of ~90% Naphthol Yellow S for microscopy (Hist.), for precipitation (of amino acids and peptides) Brilliant Green certified by the Biological Stain Commission 1-Naphthyl Red Hydrochloride, Pigment content of 85%. Petition 870210101890, dated 04 / 11 / 2021, page 47 / 165 43 / 63 Brilliant Yellow, Pigment content >50% Neutral Red powder, BioReagent, suitable for cell culture Sodium salt of bromamide acid Neutral Red, Pigment content >90% Bromobimane >97% Neutral Red certified by the Biological Stain Commission 4'-Bromo-(1,1'-biphenyl)-4-ol at 97% Nova Coccina, Pigment content of 75% Bromochlorophenol Blue, Pigment content of 95% Nigrosin certified by the Biological Stain Commission Bromocresol Green, ACS reagent, Pigment content of 95% Water-soluble Nigrosin, For use as a biological dye Sodium salt of crystalline Bromocresol Green Nile Blue A, Pigment content >75% Sodium salt of Bromocresol Green, ACS reagent, Pigment content of 90% Nile Blue A certified by the Biological Stain Commission Sodium salt of Bromocresol Green in a 0.04% wt solution in H2O; Nile Red, Technical Grade; Bromocresol Green / Methyl Red, mixed indicator solution in methanol; Indicator grade Nitrazine Yellow.Pigment content of 85% Bromocresol Green in Sultone form for microscopy (Bot., Hist., Vit.), indicator (pH 3.8-5.4) 4-Nitroazobenzene technical grade, 90% Bromocresol Purple, BioReagent, suitable for indicator, Pigment content of 90% 3-Nitrobenzaldoxime to 99% Bromocresol Purple, Technical grade 4-nitrobenzenediazonium tetrafluoroborate to 97%, Petition 870210101890, dated 04 / 11 / 2021, page 48 / 165 44 / 63 Bromocresol Purple for microscopy (Hist., Vit.), indicator (pH 5.2-6.8) 97% 4-Nitroguaiacol Sodium salt of indicator grade Bromocresol Purple, Pigment content 90% 98% hydrated 4-Nitroguaiacol Bromocresol Purple in 0.04% wt. solution in H2O 4-(4-Nitrophenylazo)resorcinol, Pigment content 90% Bromophenol Blue 97% 2-(3-Nitrophenylsulfonyl)ethanol Bromophenol Blue, ACS reagent 99% 4-Nitrophthalamide Sodium salt of Bromophenol Blue for molecular biology, for electrophoresis 97% 3-Nitrophthalimide Sodium salt of Bromophenol Blue Bromophenol 4-Nitrophthalimide 98% Sodium salt of Bromophenol Blue, Pigment content 90%, ACS reagent 3-Nitrophthalonitrile 99% Bromophenol Blue in solution at 0.04% wt in H2O 4-Nitrophthalonitrile 99% 2-(5-Bromo-2-pyridylazo)-5(diethylamino)phenol 97% Nitrotetrazolium Blue Chloride ~98% (TLC) Bromothymol Blue, ACS reagent, Reagent content 95% Nitrotetrazolium Blue Chloride powder,Sodium salt of Bromothymol Blue powder, Nitrotetrazolium Blue chloride monohydrate, Sodium salt of Bromothymol Blue, ACS reagent, 3-noradamantanamine hydrochloride at 95%, Sodium salt of Bromothymol Blue in solution at 0.04% wt in H2O, 3-noradamantananecarboxylic acid at 97%, 2-Bromo-1,1,3-trimethoxypropane at 95%, Rapid Nuclear Red. Petition 870210101890, dated 04 / 11 / 2021, page 49 / 165 45 / 63 Bromoxylenol Blue, indicator grade, 95% pigment content; Rapid Nuclear Red for microscopy (Bot., Hist.); 6-tert-Butyl-2,3-naphthalenedicarbonitrile 94% Rapid Nuclear Red; 4-tert-butylphthalic anhydride 95%; 4-tert-Butylphthalonitrile 98%; Octyl acetate >99%; Sodium salt of N-tert-butyl-α-(2-sulfophenyl)nitrone 95%; Oil Blue N, 96% pigment content; Disodium salt of sulfobromophthalein hydrate used to study hepatocyte transport functions; Oil Red O certified by the Biological Stain Commission C; Oil Red O in 0.5% solution in isopropanol; Calcein, used for the fluorometric determination of calcium and titration of calcium with EDTA. Presence of magnesium. Red Oil O in 0.5% solution in propylene glycol. Calcein AM in 4 mM solution in DMSO, >90% (HPLC). Red Oil EGN Mixed calcium and magnesium salt of calcium ionophore A23187, approximate molar ratio of 1:1 of Ca:Mg. Actual content provided on the label.Orange G for electrophoresis NA Calconcarboxylic acid Orange G certified by the Biological Stain Commission Indicator grade Calmagite Orange G certified by the Biological Stain Commission, Pigment content of 80%. Petition 870210101890, dated 04 / 11 / 2021, page 50 / 165 46 / 63 Calmagite triethanolammonium salt, Orange II sodium salt (Certified by the Biological Stain Commission), Pigment content >85%, Mounting medium, Canada balsam for microscopy, Orange OT, Pigment content 75%, Carbazole >95% (GC), Synthetic orcein, Carbol Fuchsin, Synthetic orcein, certified by the Biological Stain Commission, Carbostiril 124 99%, Orcinol monohydrate, reagent for colorimetric detection, N-hydroxysuccinimidic ester of 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfluorescein, Osmium tetroxide, Sealed Ampoule.5-Carboxyfluorescein 99% (HPLC) Oxacillin sodium salt monohydrate 6-Carboxyfluorescein ~97% (HPLC) 5-Carboxyfluorescein diacetate ~95% (HPLC) Para Red, 95% pigment content 5(6)-Carboxyfluorescein diacetate, Mixed isomers >90% (HPLC) Pararosaniline hydrochloride N-Carboxymethyl-6-(2,2-dicyanovinyl)1,2,3,4-tetrahydroquinoline >98% (HPLC) Pararosaniline acetate, 90% pigment content 5-Carboxitetramethylrhodamine Pararosaniline Base, 95% pigment content Cardiogreen polymethine pigment Pararosaniline Crystalline base Carmine powder Patent Blue VF, Pigment content of 50% Carmine certified by the Biological Stain Commission PDAM for derivatization on HPLC. Petition 870210101890, dated 04 / 11 / 2021, page 51 / 165 47 / 63 Carminic acid, 4-pentylbicyclo[2.2.2]octane-1-carboxylic acid at 99%, Celestine Blue, Pigment content 80%, Sublimed grade perylene, >99.5%, Chicago Sky Blue 6B powder, Perylene >99%, Chlorazole Black, Phenazine ethosulfate >95%, 1-Chloro-9,10-bis(phenylethinyl)anthracene at 99%, Pure phenolphthalein, meets the analytical specification of Ph Eur., BP, 98-101% (calc.(for the dry substance) 2-Chloro-5-methylaniline at 99% Phenolphthalein, ACS reagent 2'-Chloro-5'-methylbenzanilide at 97% Phenolphthalein in solution at 0.5% wt in ethanol:water (1:1) 5-Chloro-2-methylindole at 97% Tetrasodium salt of phenolphthalein bisphosphate at ~95% 2-Chloro-6-nitrobenzaldehyde at 97% Phenol Red powder, BioReagent, suitable for cell culture Indicator grade Chlorophenol Red Phenol Red, ACS reagent Chlorophyll a from algae Anacystis nidulans Sodium salt of Phenol Red powder, BioReagent, suitable for cell culture, suitable for insect cell culture Spinach Chlorophyll b >90% (HPLC), <0.5% Chlorophyll a Sodium salt of Phenol Red Salt of 4-chloro-7-sulfobenzofurazane ammonium sodium salt of Phenol Red, ACS reagent, 90% pigment content. Chromeazurol B Phenol Red in 0.5% solution, liquid, filtered under sterile conditions. Petition 870210101890, dated 04 / 11 / 2021, page 52 / 165 48 / 63 BioReagent, suitable for cell culture; Chromeazurol S, 50% pigment content; Phenosafranin, 80% pigment content; Chromotrope 2R suitable for staining; Modified Gomori Trichrome; 3-Phenoxyphthalonitrile 98%; Chromotrope FB, 50% pigment content; 4-Phenoxyphthalonitrile 98%; Chrysin 97%; N-[5-(phenylamino)-2,4-pentadienylidene]aniline monohydrochloride 98%; Chrysoidin G for microscopy (Bact., Bot., Vit.)) 4-(Phenylazo)diphenylamine at 97% Chrysophenine, Pigment content 65% 4-Phenylazophenol at 98% Brilliant Yellow Cibacron 3G-P Anhydride 1-phenyl-2,3-naphthalenedicarboxylic acid at 98% Clobetasone butyrate >98% Internal salt of 5-phenyl-2-[2[[5-phenyl-3-(3-sulfopropyl)-2(3H)-benzoxazolylidene]methyl]-1-butenyl]-3-(3-sulfopropyl)benzoxazolium hydroxide, sodium salt, Pigment content 90% Collodion solution for microscopy, 2% in amyl acetate 2-(Phenylsulfonyl)ethanol at 97% Congo Red certified by the Biological Stain Commission, BioXtra Phloroglucinol, Used for Detect the presence of wood fiber. Congo Red, Pigment content >35% Fluorescent pigment Phloxine B antibacterial Coomassie Violet R200, Pigment content 50% Phloxine B, Pigment content >80%, certified by the Biological Stain Commission. Petition 870210101890, dated 04 / 11 / 2021, p. 53 / 165 49 / 63 Éster tetrametílico da coproporfirina I >90% (HPLC) Brometo de pinacianol, Teor de pigmento de 95 % Cumarina 6 a 98% Cloreto de pinacianol Cumarina 7 a 98% Poli(1 -metoxi-4-(O-Vermelho disperso 1 ))-2,5-fenilenovinileno Cumarina 314, Teor de pigmento de 97 % Ponceau S BioReagent, adequado para eletroforese Cumarina 334, Teor de pigmento de 99 % Ponceau S para microscopia (Hist.Coumarin 343, Pigment content 97% Ponceau S, Pigment content 75% o-Cresolphthalein indicator grade Ponceau S in solution BioReagent, suitable for electrophoresis, 0.1% (w / v) in acetic acid 5% o-Cresolphthalein Complexone powder Ponceau BS, Pigment content ~60% m-Cresol Purple indicator grade, Pigment content 90% Ponceau SS, Pigment content 80% Sodium salt of m-Cresol Purple, Pigment content 90% Xylidene Ponceau, Pigment content >60% Cresol Red indicator grade, Pigment content 95% Potassium Indigotetrasulfonate, Pigment content 85% Sodium salt of Cresol Red indicator grade Reagent remover of Ozone, Potassium Indigotrisulfonate, Cresyl Acetate, Violet certified by the Biological Stain Commission, Procion® Red MX-5B, Pigment content of 40%, Crocein Scarlet 7B, Proflavin hemisulfate salt hydrate powder, Croconic acid at 98%, propidium iodide >94.0% (HPLC). Petition 870210101890, dated 04 / 11 / 2021, p. 54 / 165 50 / 63 Analytical standard Crotonaldehyde-2,4-dinitrophenylhydrazone, for environmental analysis Propional iodide in solution (1.0 mg / mL in water) Crystal Ponceau 6R Analytical standard Propionaldehyde-2,4-dinitrophenylhydrazone, for environmental analysis Crystal Violet, Pigment content >90% Soluble Prussian Blue for microscopy Crystal Violet, ACS reagent, >90.0% anhydrous base Purpurin, Pigment content 90% Crystal Violet certified by the Biological Stain Commission N-hydroxysuccinimidic ester of 1-pyrenebutyric acid 95% Crystal Violet in solution 1%, aqueous solution 3,4-Pyridinodicarbonitrile 96% Crystal Violet Lactone, Pigment content 97% 3,4-Pyridinodicarboxamide 98% Curcumin from Curcuma longa (Saffron), powder 1-(2-Pyridylazo)-2-naphthol of indicator grade Internal salt of N-[3-Cyano-3-[4-(dicyanomethyl)phenyl]-2-propenylidene]-Netyl-ethaniminoate 4-(2-Pyridylazo)resorcinol at 96% Hydroxysuccinimidic acid ester chloride of 9-Cyano-N,N,N'-triethylpyroninoN'-caproic acid >85% (HPLC) Monosodium salt of 4-(2-pyridylazo)resorcinol hydrate N4-(maleimidoethyl)piperazide chloride of 9-cyano-N,N,N'-triethylpyroninoN'-caproic acid >80% (HPLC) Disodium salt of 3-(2-pyridyl)-5,6di(2-furyl)-1,2,4-triazino-5',5''-disulfonic acid Cyclohexanone 2,4-dinitrophenylhydrazone >99% Sodium salt of 3-(2-pyridyl)-5,6diphenyl-1,2,4-triazino-4',4''-disulfonic acid BioXtra, Petition 870210101890, dated 04 / 11 / 2021, page 55 / 165 51 / 63 D Pyrocatechol Violet suitable for DAPI indicator, dilactate >98% (HPLC) Pyrogallol Red Darrow Red certified by the Biological Stain Commission, Pigment content >65% Pyronine Y for electrophoresis NA o-Dianisidine Dihydrochloride >95% Pyronine B, Pigment content >30% o-Dianisidine Dihydrochloride, Suitable for use in glucose determination Pyronine B certified by the Biological Stain Commission, Pigment content of 40% o-Dianisidine Dihydrochloride for enzymatic, spectrophotometric determination, 5 mg vial Pyronine Y for microscopy (Bot., Fl., Hist.) 4,5-Dibromobenzene-1,2-diol at 90%, more technical Pyronine Y certified by the Biological Stain Commission, Pigment Theor of 50 % 4',5'-Dibromofluorescein, Pigment Theor of 95 % Q 2,5-63-methobromol-Dibromol-Diol Qdot 525 97% 3,6-Dibuthose-1,2-Benzenodicarbonitrile Qdot 565 2,5-Dibutose-4-(4morpholynyl)benzenodiazone Tetrafluoroborate, 95 % Qdot 605 Pigment Theor 1,4-Dibutoxy-2,3-naphthalenodicarbonitrile at 99% Qdot 655 4-(Dibutylamino)benzaldehyde at 98% Qdot 705. Petition 870210101890, dated 04 / 11 / 2021, p. 56 / 165 52 / 63 Internal salt of 4-(2-(6(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3sulfopropyl)pyridinium hydroxide >95% (HPLC), solid Qdot 800 N,N-Dibutylaniline at 97% Quinaldine Red, Pigment content 95% 1,5-Naphthalenedisulfonate of 2,4-dichlorobenzenediazonium hydrate Quinoline Yellow for microscopy (Hist.), mixture of mono- and disulfonic acid sodium salts 3,6-Dichloro-1,2-benzenedithiol at 95% Quinoline Yellow, Pigment content 95% 1,2-Dichloro-4,5-dinitrobenzene at 98% Quinoline Yellow, Mixture of mono- and disulfonic acids of Quinoline Yellow 2',7'-Dichlorofluorescein at ~90% (TLC), Crystalline R 2',7'-Dichlorofluorescein, ACS reagent Black Reactive 5, Pigment content >50% Sodium salt of 2,6-dichloroindophenol hydrate suitable for vitamin C determination, BioReagent Blue Reactive 4, Pigment content of 35% 2,6-Dichloroquinone-4-chloroimide Practical grade Green Reactive 19 6-(2,2-Dicyanovinyl)-N-(2-hydroxyethyl)-1,2,3,4-tetrahydroquinoline Orange Reactive 16,Pigment content >70% trans-4-(Diethylamino)cinnamaldehyde at 98% Reactive Red 120 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin >95% (HPLC), solid Reichardt Pigment, Pigment content of 90% 1,1'-Diethyl-2,2'-carbocyanine iodide at 97% Anthraquinone Pigment Remazol Brilliant Blue R 1,1'-diethyl-2,2'-cyanine iodide at 97% Renin 1 Substrate, Petition 870210101890, dated 04 / 11 / 2021, page 57 / 165 53 / 63 lodeto de 1,1'-diethyl-4,4'-cianina Sal sódico da resazurina certificado pela Biological Stain Commission Sal de oxalato de N,N-diethyl-p- fenilenodiamine Resorcinol BioXtra, >99% 3,4-Piridinodicarboxilato de dietila a 97% Resorufina, Teor de pigmento de 95 % lodeto de 3,3'-diethylthiacianina, Teor de pigmento de ~97 % Rodamine 6G, Teor de pigmento de 99 % Di-hidroetídeo >95% Rodamine 6G, Teor de pigmento de ~95 % Di-hidrorrodamine 123 >95% Perclorato de rodamine 6G, Teor de pigmento de 99 % 2',4'-Di-hidroxi-3'-methylacetofenona de grau técnico, 90% Rhodamine B >95% (HPLC) 1,3-Di-hidroxinaphtaleno >99%, crystalline Chloreto de rhodamine 110, Pigment content >88 % 1,4-Di-hidroxi-2,3-naphtalenodicarbonitrila a 97% Fluorescent dye specific for mitochondria Rhodamine 123 4,7-Di-hidroxi-1,10-phenanthroline, Pigment content >30% Rhodamine B base, Pigment content 97% 1,3-Diiminobenz[f]isoindoline at 95% Isothiocyanate of Rhodamine B-Dextran com p. mol.Average of ~10,000 1,3-Diiminoisoindoline at 97% Mixed isomers of Rhodamine B isothiocyanate Technical grade 3,3'-Dimethoxybenzidine dihydrochloride Rhodamine Blue A complex of Nile Blue and Rhodamine B, Pigment content of 75% (2,5-Dimethoxyphenyl)acetyl chloride at 99% RIM-1 >90% (HPLC) 2,4-Dimethoxytoluene at 99% Rose Bengal, Pigment content of 95%. Petition 870210101890, dated 04 / 11 / 2021, page 58 / 165 54 / 63 4-(Dimethylamino)benzaldehyde suitable for histochemical demonstration of tetrazolium nitroblue reduction in neutrophils. Sodium salt of Rose Bengal, Pigment content >85%. Chromogenic reagent 4-(dimethylamino)cinnamaldehyde for indoles and flavonoids. Rose Bengal diacetate. 4-Dimethylamino-2-methylazobenzene. Rose Bengal lactone at 95%. 2-[4-(dimethylamino)styryl]-1-ethylpyridinium iodide >99% (HPLC), solid. p-Rosolic acid, Pigment content 85%. 2-[4-(dimethylamino)styryl]-N-methylbenzoxazolium perchlorate. Rubrene powder. N,N-Dimethyl-4,4'-azodianiline at 97%. Ruthenium red, Technical grade. Dinitrate of N,N'-dimethyl-9,9'-biacridinium used as a chemiluminescent reagent. Ruthenium Red for microscopy, >85% (calc.For the dry substance, AT) N,N-Dimethylindoaniline, Pigment content 97% S 3,3-Dimethyl-2-methylene-1-phenylindoline Impure saffron, source of crocetin and crocein N,N-Dimethyl-1-naphthylamine >98.0% (GC) Safranin O, Pigment content >85% N,N-Dimethyl-4-nitrosoaniline at 97% Safranin O certified by the Biological Stain Commission N,N-Dimethyl-p-phenylenediamine dihydrochloride suitable for peroxidase test, >99.0% (titration) Safranin O for microscopy (Bact., Bot., Hist.), indicator (pH 0.3-1.0) 1,4-Dimethylpyridinium iodide at 99% Schiff fuchsin-sulfite reagent suitable for glycoprotein detection. Petition 870210101890, dated 04 / 11 / 2021, page 59 / 165 55 / 63 1,4-dimethylpyridinium p-toluenesulinate 98% Scopoletin >99% 3,5-Dinitroaniline 97% Silver diethyldithiocarbamate 4,4'-Dinitro-2-biphenylamine 98% Silver Reinforcer Kit 10-(2',4'-Dinitroenylazol)-9-enantrole Silver Reinforcer Solution A 3,5-Dinitrosalicylic acid used in the colorimetric determination of reducing sugars Silver Reinforcer Solution B 1,1'-dioctadecyl-4,4'-bipyridinium dibromide 97% BioXtra silver nitrate, >99% (titration) 3,3'-dioctadecyloxacarbocyanine perchlorate ReagentPlus® silver nitrate, >99.0% (titration) Internal salt 4-(2-[6-(dioctylamino)-2-nitalenyl]ethenyl)-1-(3-sulfopropyl)pyridinium >95% (HPLC), solid Silver proteinate ~8% based on Ag 1,3-Dienylacetone-p-tosylhydrazone 98% Practical grade Blue 38 solvent 2-Dienylacetyl-1,3-indandione-1-hydrazone 98% Blue 59 solvent, 98% pigment content 1,2-Dienylindole 94% Green 3 solventPigment content of 95% N-(dienylmethylene)glycine tert-butyl ester at 98% Stains-All at ~95% 2,6-Dienyl-4H-thiopyran-4-one at 96% trans-4-Stilbenomethanol Direct Blue 71, Pigment content of 50% Sudan I, Pigment content >95% Direct Blue 15 suitable for the Histopaque® system, suitable for viability studies of rat liver cells treated with collagenase Sudan II, Pigment content of 90%, Petition 870210101890, dated 04 / 11 / 2021, pp. 60 / 165 56 / 63 Direct Red 23, Pigment content of 30% Sudan III, Technical grade Direct Red 80, Pigment content of 25% Sudan III certified by the Biological Stain Commission, BioXtra Direct Red 81, Pigment content of 50% Sudan IV certified by the Biological Stain Commission, BioXtra Direct Yellow 27 Sudan IV, Pigment content >80% Disperse Black 9, Pigment content of 97% Sudan Black B certified by the Biological Stain Commission Disperse Blue 1, Pigment content of 30% Sudan Blue II, Pigment content of 98% Disperse Blue 3, Pigment content of 20% Sudan Orange G, Pigment content of 85% Disperse Blue 14, Pigment content of 97% Sudan Red 7B for microscopy (Bot., Hist.Dispersed Orange 1, Pigment content ~15% Sudan Red 7B, Pigment content 95% Dispersed Orange 13, Pigment content 90% Azochromotrope Sulfanilic acid >80% Dispersed Orange 13, Pigment content 15% Technical grade 2-sulfobenzoic acid cyclic anhydride, 90% Dispersed Yellow 3, Pigment content 30% Sodium and calcium salt of sulfochlorophenol S Dithizone, Practical Grade Sulforhodamine 101 Dithizone, ACS reagent, >85.0% Sodium salt of sulforhodamine B, Technical Grade E Sunset Yellow FCF, Pigment content 90%. Petition 870210101890, dated 04 / 11 / 2021, page 61 / 165 57 / 63 Eosin B certified by the Biological Stain Commission, 90% pigment content. Green alternative to RNA gel dye SYBR® Green II 10,000 χ in DMSO. Eosin B for microscopy (Fl., Hist.), adsorption indicator and fluorescent. Green alternative to nucleic acid gel dye SYBR® Green I 10.000 χ in DMSO Eosin B, Pigment content 95% SynaptoGreen™ C4 >95% (HPLC), solid Eosin Y, Pigment content ~99% SynaptoRed™ C2 >95% (HPLC), solid Eosin Y Syringaldazine 99% Eosin Y disodium salt, Pigment content >85% Eosin Y disodium salt certified by the Biological Stain Commission Tannic acid source: Natural Chinese oak galls Eosin Y in 5% wt solution in H2O Tetrabromophenol Blue, Pigment content 85% Eriochrome Black® T, ACS reagent (indicator grade) Tetrabromophenol Blue sodium salt, Pigment content 85% Erioglaucine disodium salt Potassium salt of ethyl ester of 3',3,5',5-tetrabromophenolphthalein indicator grade Erythrosine B, Pigment content >95% 3,4,5,6-Tetrabromophenolsulfonphthalein, Pigment content 95% Erythrosine B certified by the Biological Stain Commission, Pigment content 90% Cyclic anhydride of tetrabromo-2-sulfobenzoic acid Extra-blue Erythrosine for microscopy (Bact., Hist.)), adsorption and fluorescent indicator Bis(3,6-dichloro-1,2-benzenedithiolate) tetrabutylammonium nickelate at 98%. Petition 870210101890, dated 04 / 11 / 2021, page 62 / 165 58 / 63 Erythrosine extra-blue certified by the Biological Stain Commission Bis(4-methyl-1,2-benzenedithiolate) tetrabutylammonium nickelate 98% Yellowish Erythrosine Mixture Tetrachlorophthalonitrile 98% Ethidium bromide ~95% (HPLC) Tetrachromic dye (MacNeal) Ethidium bromide monoazide >95% (HPLC), solid 2,3,6,7-Tetrahydro-8-hydroxy-1H,5Hbenzo[ij]quinolizino-9-carboxaldehyde 98% Ethidium I homodimer in solution >95%, 2 mM in DMSO 1,2,3,4-Tetrahydro-2,2,4,7-tetramethylquinoline 97% iodide 5-[3-ethoxy-4-(3-ethyl-5-methyl2(3H)-benzothiazolylidene)-2-butenylidene]-3-ethyl-2-[(3-ethyl-4,5-diphenyl2(3H)-thiazolylidene)methyl]-4,5-dihydro-4oxothiazolium, Pigment content 95% 3',3,5',5''-Tetraiodophenolsulfonephthalein, 90% pigment content 99% 3-Ethoxy-4-methoxybenzaldehyde 98% 1,2,3,3-tetramethyl-3H-indolium lodeide Internal hydroxide salt 2-[3-(3-ethyl2(3H)-benzothiazolylidene)-2-methyl-1propenyl]-3-[3-(sulfo- oxy)butyl]benzothiazolium, Pigment content of 90% 2,2,4,4-Tetramethyl-3-pentanoneimine 95% 5-Ethyl-5,6-dihydro-3,8-dinitro-6-phenyl-6-phenantridinol 97% N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride >95%, powder Ethyl 3,5-Dimethyl-2-pyrrolocarboxylate 98% Tetramethylrhodamine-5-isothiocyanate Ethyleosin certified by the Biological Stain Commission Tetramethylrhodamine methyl ester perchlorate >95% Ethyl 4'-Hydroxy-4-biphenylcarboxylate 98% Tetrazolium tetranitroblue chloride Petition 870210101890, dated 04 / 11 / 2021, page 63 / 165 59 / 63 3-Ethyl-2-methylbenzothiazolium iodide Tetrazolium Blue Chloride used in the colorimetric determination of reducing compounds Sodium salt of Ethyl Orange of indicator grade, Pigment content 90% Tetrazolium Violet 2-(Ethylthio)benzothiazole at 97% Potassium salt of TFLZn >90% (TLC) 2-Ethyl-2-thiopeseudourea hydrobromide at 98% TFLZn-AM >90% (TLC) N-Ethyl-o-toluidine at 97% Thiazole Orange, Pigment content ~90% Triarylmethane cationic pigment Ethyl Violet Thiazolyl Bromide Tetrazolium Blue at 98% Ethylviologen Diperchlorate at 98% Practical grade Thioflavin S Evans Blue, Pigment content >75 Thioflavin T used as a dye for amyloid F Thioflavin acetate salt certified by the Biological Stain Commission, Pigment content >85% Fast Black Salt K, practical grade hemi(zinc chloride) salt Thymol Blue, ACS reagent Fast Blue Salt BB, hemi(zinc chloride) salt, Pigment content >80% Sodium salt of Thymol Blue, ACS reagentPigment content of 95% Fast Blue BB Salt, zinc chloride hemi(chloride) salt for microscopy (Hist.) Thymolphthalein, ACS reagent, Pigment content of 95% Fast Blue RR Thymoquinone >98% Fast Blue RR crystalline o-Toluidine >97%, powder Fast Blue B Salt, Pigment content of ~95% Toluidine Blue O, Technical grade, Petition 870210101890, dated 04 / 11 / 2021, p. 64 / 165 60 / 63 Double salt of zinc chloride Fast Corinth V, Pigment content of 90% Toluidine Blue Certified by the Biological Stain Commission Black Blue Salt Fast R Toluidine Red, Pigment content of 70% Diazonium dye Garnet Sulfate Salt Fast 3,4,5-Trihydroxybenzamide at 98% Base Fast Garnet GBC at 97% Technical grade Tris(4-nitrophenyl)amine Fast Green FCF, Pigment content >85% Trypan Blue, Pigment content of 60% Fast Green FCF Certified by the Biological Stain Commission Trypan Blue Powder, BioReagent, suitable for cell culture Fast Red B Tetrafluoroborate Salt, Pigment content of 95% Trypan Blue in 0.4% solution, liquid, filtered under sterile conditions, suitable for cell culture Fast Red ITR U Fast Red RC Salt Uniblue A Sodium Salt Fast Red Violet LB V Salt Rapid Violet Red LB, Pigment content >90% Environmental standard Valeraldehyde-2,4-dinitrophenylhydrazone,99% Fatty Brown B Vanillin azine at 99% Indicator Ferroin in solution at 0.1% wt in H2O Salt of Variamine Blue RT FIM-1 Victoria Blue R, Pigment content of 80% FIM-1 Diacetate Pure Victoria Blue BO, Pigment content of 90% Sodium salt of fluorescein used as a fluorescent marker Violamin R, Pigment content of 60%, Petition 870210101890, dated 04 / 11 / 2021, pp. 65 / 165 61 / 63 Fluorescein N-hydroxysuccinimidic ester-5-EX W Fluoresceinamine, isomer I Wright's stain suitable for blood staining Fluorescein diacetate used as a stain for cell viability Wright's stain certified by the Biological Stain Commission Fluorescein (free acid), 95% pigment content Wright's stain in solution for microscopy 5(6)-Isothiocyanate Fluorescein >90% (HPLC) X Isothiocyanate Fluorescein Isomer I suitable for protein labeling, >90% (HPLC), powder p-Xylene-bis(N-pyridinium bromide) >95% (TLC) Isothiocyanate Fluorescein Isomer I >97.5% (HPLC) Xylene Cyanol FF, Pigment content >75% Isothiocyanate Fluorescein Isomer I Celite® suitable for fluorescent labeling techniques Xylene Cyanol FF for molecular biology, BioReagent Sodium Salt of Fluorescein - CAPS solution for fluorescence, >95.0% (HPLC) Xylenol Blue indicator grade,Pigment content 90% Fluorescent Brightener 28 used as a colorant and brightening agent Tetrasodium salt of Xylenol Orange, ACS reagent Fluorinert™ FC-40 Xylidyl Blue I Disodium salt of 4-formylbenzene-1,3-disulfonic acid hydrate at 97% Z Fura 2-AM >95% (HPLC) Zincon sodium salt, Pigment content >75% Fura-2 LeakRes (AM) >85% Zincon >95% (HPLC), solid, Petition 870210101890, dated 04 / 11 / 2021, page 66 / 165 62 / 63 Fusidic acid, ethyl ester of zinc >95% (HPLC), solid Plasmocorinth B, pigment content 60%

[0064] When ranges are provided, the extremes are included. Furthermore, it should be understood that, unless otherwise indicated, or if otherwise evident from the context and / or the understanding of a person skilled in the art, the values ​​expressed as ranges may assume any specific value within the ranges indicated in different embodiments of the invention, down to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It should also be understood that, unless otherwise indicated, or if otherwise evident from the context and / or the understanding of a person skilled in the art, the values ​​expressed as ranges may assume any sub-range within the range provided, wherein the extremes of the sub-range are expressed to the same degree of precision as the tenth of the unit of the lower limit of the range.

[0065] The term including, whenever used in this document, is intended to indicate the presence of referenced features, integers, steps, components, but not to exclude the presence or addition of one or more other features, integers, steps, components, or their groups.

[0066] Disclosure is evidently by no means restricted to the forms described and a person skilled in the art will foresee many possibilities for modifications thereto without departing from the basic idea of ​​disclosure as defined in the appended claims.

[0067] The modalities described above are obviously combinable.

[0068] The following dependent claims establish particular modalities of disclosure. SEQUENCE LISTING SEQ- ID. No. 1: Folic acid-DRDDQAAWFSQY SEQ-ID. No. 2: KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK-DRDDQAAWFSQY SEQ-ID. No. 3: YQSFWAAQDDRD-KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK Petição 870210101890, de 04 / 11 / 2021, pág. 67 / 165 63 / 63 REFERÊNCIAS [1] Wang, A. Z., Langer, R., e Farokhzad, O. C., Nanoparticle Delivery of Cancer Drugs, Annual Review of Medicine. 2012, 63, 185-198. [2] Hong, M.-S., Lim, S.-J., Lee, M.-K., Kim, Y. B., e Kim, C.-K., Prolonged Blood Circulation of Methotrexate by Modulation of Liposomal Composition, Drug Delivery. 2001,8, 231-237. [3] Laouini, A., Jaafar-Maalej, C., Limayem-Blouza, I., Sfar, S., Charcosset, C., e Fessi, H., Preparation, Characterization and Applications of Liposomes: State of the Art, Journal of Colloid Science and Biotechnology. 2012, 1, 147-168. [4] Bangham, A. D., Standish, M. M., e Watkins, J. C., Diffusion of univalent ions across the lamellae of swollen phospholipids, Journal of Molecular Biology. 1965, 13, 238-IN27. [5] Batzri, S. e Korn, E. D., Single bilayer liposomes prepared without sonication, Biochimica et Biophysica Acta (BBA) - Biomembranes. 1973, 298, 1015-1019. [6] Naeff, R., Feasibility of topical liposome drugs produced on an industrial scale, Advanced Drug Delivery Reviews. 1996, 18, 343-347. [7] Sur, S., Fries, A. C., Kinzler, K. W., Zhou, S., e Vogelstein, B., Remote loading of preencapsulated drugs into stealth liposomes, Proceedings of the National Academy of Sciences of the United States of America. 2014, 111, 2283-2288. [8] Gubernator, J., Active methods of drug loading into liposomes: Recent strategies for stable drug entrapment and increased in vivo activity. Vol. 8. 2011. 565-80. [9] Duong, A. D., Collier, M. A., Bachelder, E. M., Wyslouzil, B. E., e Ainslie, K. M., One Step Encapsulation of Small Molecule Drugs in Liposomes via ElectrosprayRemote Loading, Mol Pharm. 2016, 13, 92-9.

[10] Jaafar-Maalej, C., Diab, R., Andrieu, V., Elaissari, A., e Fessi, H., Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation, J Liposome Res. 2010, 20, 228-43.

[11] Pons, M., Foradada, M., e Estelrich, J., Liposomes obtained by the ethanol injection method, International Journal of Pharmaceutics. 1993, 95, 51-56.

Claims

1. Method for encapsulating an active ingredient in a liposome, characterized by consisting of the following sequential steps: V Prepare an ethanolic phase by mixing hydrophobic phospholipid molecules and a steroid with ethanol; V Prepare an aqueous phase with an active ingredient and a targeting agent in a buffer solution; V Obtain liposomes by injecting the ethanolic phase into the aqueous phase, at a temperature of 50°C to 80°C, wherein the volume ratio of the ethanolic / aqueous phases is between 1:1 and 3:2; V Remove the ethanol; V Remove the remaining free active ingredient in a suitable manner, namely by tangential flow filtration; V Optionally, the step of diluting the liposomal dispersion 1 to 10 times in a more dilute aqueous phase wherein the targeting agent is a peptide that is conjugated to a liposomal component or incorporated into the lipid membrane; and in which the concentration of ethanol, relative to the initial aqueous volume, is between 40 and 60%.

2. Method according to claim 1, characterized in that the removal of ethanol is carried out by evaporation or tangential flow filtration.

3. Method according to claim 1, characterized in that the steroid is cholesterol.

4. Method according to claim 1, characterized in that the cholesterol is cholesteryl hemisuccinate.

5. Method, according to any of the preceding claims, characterized in that the ethanolic phase is injected at a rate of approximately 2-4 mL / minute. Petition 870240100229, dated 11 / 25 / 2024, page 21 / 29 2 / 3 6. A method, according to any of the preceding claims, characterized in that the ethanol concentration, with respect to the initial aqueous volume, is 50%.

7. A method, according to any of the preceding claims, characterized in that the temperature is 60°C or 70°C.

8. A method, according to any of the preceding claims, characterized in that the active ingredient is a polycharged molecule containing at least one negative charge at a pH of about 4 to about 7.

9. A method, according to any of the preceding claims, characterized in that the active ingredient is a drug, an imaging agent, or a therapeutic agent, in particular, an anticancer drug, an antirheumatic drug, a drug against neurodegenerative diseases, an antioxidant drug, an anti-inflammatory drug, an antipyretic drug, an antibiotic drug, an antiviral drug, an analgesic drug, or combinations thereof, in particular, the imaging agent being a pigment.

10. A method, according to any of the preceding claims, characterized in that the injection step is carried out under agitation.

11. A method, according to any of the preceding claims, characterized in that the targeting agent is at least one peptide selected from the following list with a degree of identity of at least 90% to the following sequences: SEQ ID. No. 1, SEQ ID. No. 2, SEQ ID. No. 3, or mixtures thereof.

12. A method, according to any of the preceding claims, characterized in that the aqueous phase is a phosphate-buffered saline solution, PBS.

13. A method, according to any of the preceding claims, characterized in that the ethanolic phase comprises anionic, neutral or cationic phospholipids.

14. Method, according to any of the preceding claims, characterized in that the phospholipids of the ethanolic phase are selected from a list consisting of: phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, Petition 870240100229, dated 11 / 25 / 2024, page 22 / 29 3 / 3 phosphatidylglycerols and / or their derivatives or mixtures thereof, in particular, 1,2-dioleoylsn-glycero-3-phosphoethanolamine.

15. A method, according to any of the preceding claims, characterized in that the ethanolic phase further comprises a stealth agent, a targeting agent, or a mixture thereof.

16. A method, according to any of the preceding claims, characterized in that the stealth agent is polyethylene glycol, PEG, or gangliosides.

17. A method, according to any of the preceding claims, characterized in that polyethylene glycol, PEG, is linked to a phospholipid, in particular, distearolphosphatidylethanolamine.

18. A method, according to any of the preceding claims, characterized in that the targeting agent is incorporated into the lipid membrane.

19. A method, according to any of the preceding claims, characterized in that the active ingredient is an imaging or therapeutic agent.

20. A method, according to any of the preceding claims, characterized in that the imaging or therapeutic agent is hydrophobic or hydrophilic.

21. A method, according to any of the preceding claims, characterized in that the imaging agent is a pigment.

22. Method according to claim 21, characterized in that the active ingredient is methotrexate, doxorubicin, or mixtures thereof.