KR101612194B1 - Composition for drug delivery comprising Liposome Encapsulated Nanoparticle of drug combined with Albumin - Google Patents

Abstract

The present invention relates to a composition for drug delivery comprising a liposome encapsulated with nanoparticles containing a drug bound to albumin, and a method for producing the same.
The drug delivery compositions of the present invention provide uniform hydrodynamic size, high packing efficiency and loading capacity and have a higher colloidal stability than the simple complex of paclitaxel and albumin similar to Abraxane ( ^R ) . In addition, since the composition for drug delivery exhibits better in vitro and in vivo behavior such as better cell uptake, cytotoxicity, and pharmacokinetic characteristics than paclitaxel-albumin simple complex and paclitaxel dissolved in crepolm / ethanol, the paclitaxel- May be usefully employed as a means for the delivery of soluble drugs.

Description

[0001] The present invention relates to a composition for drug delivery comprising a liposome encapsulated with nanoparticles containing a drug bound to an albumin,

The present invention relates to a composition for drug delivery comprising a liposome encapsulated with nanoparticles containing a drug bound to albumin, and a method for producing the same.

A poorly soluble drug means a drug that is insoluble in water because it contains a structurally hydrophobic part of the compound, and its practicality is often limited due to poor availability. For example, about 41% or more of new drug products are being abandoned due to poor availability, and about one-third or more of the drugs listed in the US Pharmacopeia are classified as poorly soluble drugs. In order to use such an insoluble drug, an additional substance for solving poor solubility has to be added. However, there have been reported many cases in which use is limited due to the toxicity of the added substance. For example, emulsification using an emulsifying agent and trapping using liposomes are widely used for water-insoluble materials in general, and their use is limited due to the incorporation of foreign substances not derived from the human body and physical instability Weiss RB et al., Journal of clinical Oncology.1990).

As a representative example of such a poorly soluble drug, paclitaxel is an anticancer substance present in nature and is a drug containing a diterpenoid derivative extracted from the skin of Taxus brevifolia, The efficacy is known for a variety of cancers. It is also known to have anti-mitotic activity that facilitates the conversion of tubulin combinations into stable microtubules. The paclitaxel basically has an alkaloid structure composed of a taxane ring and an ester side chain and shows poor solubility.

Paclitaxel inhibits the depolymerization of macromolecularized microtubules and inhibits cell replication at the delayed G ₂ / M phase of the cell cycle cycle. Thus, paclitaxel has shown excellent potential as an antitumor drug, but its low solubility in water has limited its widespread use in the field of cancer therapy.

In order to solve the poor solubility of paclitaxel which inhibits the use of paclitaxel as an anticancer agent, it has been dissolved in ethanol, but recently it has been used as an injectable drug in combination with albumin in order to increase the delivery efficiency.

There is also known a method for use in systemic administration by improving the water solubility of paclitaxel using a solvent called Cremorphor EL, which is a mixture of polyoxyethylated castor oil and absolute ethanol . The formulation commercialized in this form is Taxol ( ^R ). However, clinically it has been reported that overdose of these solvents causes side effects such as cardiac toxicity, neurotoxicity, neuropathy and hypersensitivity (Onetto, N. et al., J. Natl. Cancer Inst. Monogr., 1993, 15 1997, 54: 566; Gregory R. et al., Clin. Pharm., 1993, 12: 401).

Abraxane ^® is formulated in the form of PTX nanoparticles bound to human serum albumin in order to overcome the disadvantages of taxol such as side effects caused by the above-mentioned solvents, Experiments have shown enhanced tumor response, but did not improve the serum half-life, or pharmacokinetic properties, of paclitaxel. This is due to the low colloidal stability of the ascorbic acid in the blood circulation process. That is, when diluted with a large volume of blood by intravenous injection of abraxan, ablacic acid rapidly degrades to release paclitaxel, resulting in a nonspecific biodistribution similar to free paclitaxel, which is difficult to deliver to targeted lesions (Hyodo K , et al, biological & pharmaceutical Bulletin. Therefore, it is still required to develop a method for stably solubilizing paclitaxel and improving the bioavailability without using a solvent.

Accordingly, the present inventors have made intensive studies in order to find a drug carrier capable of minimizing adverse effects when a poorly soluble drug is systemically administered. As a result, it has been found that an insoluble drug, which is naturally present in the body and is water- It has been confirmed that the nanoparticles are formed and the nanoparticles are encapsulated in liposomes to formulate the drug, thereby alleviating the side effects of the conventional drug delivery system and improving the pharmacokinetic properties of the poorly soluble drug.

It is an object of the present invention to provide a composition for drug delivery, which comprises a liposome encapsulated with nanoparticles containing an albumin-bound drug.

It is another object of the present invention to provide a method for producing nanoparticles comprising: a first step of preparing nanoparticles containing a drug bound to albumin; And a second step of enclosing the nanoparticles prepared in the first step into a liposome, wherein the nanoparticles comprise a drug bound to albumin.

In order to achieve the above object, the present invention provides a composition for drug delivery improved in pharmacokinetic characteristics comprising a liposome encapsulated with nanoparticles containing a drug bound with albumin.

The composition for drug delivery of the present invention does not require a solvent such as CREMOPOL EL used for the solubilization of a poorly soluble drug so that it does not show adverse effects that may appear from a toxic solvent, By enclosing the nanoparticles bound to protein albumin in the liposome, the pharmacokinetic properties of the drug are superior to those of albumin or liposome as a carrier. For example, it is possible to provide a drug delivery system capable of overcoming the pharmacodynamic disadvantages in abraic acid, which has improved colloid stability and drug solubility, has an extended release profile at physiological pH values, and a high cell uptake rate Feature.

The "albumin" of the present invention is one of the proteins constituting the basic substance of a cell, and is the smallest molecular weight protein among the simple proteins existing in a natural state. It is the predominant protein found in plasma as water-soluble. The albumin can also be extracted from egg white (ovalbumin), bovine serum, human serum or cereal, and is obtained from a transformant prepared to express a polypeptide having the same amino acid sequence as naturally occurring albumin One recombinant albumin may be used, but is not limited thereto.

The albumin may bind to various ligands such as cations such as water, calcium, sodium, and potassium, fatty acids, hormones, bilirubin, and drugs to control the colloid osmotic pressure of blood.

"Drug" of the present invention may be a water-insoluble drug which is alcohol-soluble.

The medicament of the present invention is not limited to water-soluble and alcohol-soluble substances, for example, paclitaxel, nifedipine, propofol, diazepam, estradiol, cyclosporine, ritonavir, saquinavir, biphenyl dimethyl dicarboxylate (BDD), coenzyme Q10 (coenzyme Q10; (UDCA), ilaprazole, imatinib mesilate, acyclovir, allopurinol, amiodarone, azathioprine (CoQ10), Ursodeoxycholic acid (UDCA) azathioprine, benazepril, calcitriol, candesartan, eprosartan, carbidopa / levidopa), clarithromycin A compound selected from the group consisting of clarithromycin, clozapine, desmopressin acetate, diclofenac, enalapril, famotidine, felodipine, fenofibrate, Fentanyl, fexofenadine, fosinopril, furosemide, glyburide, hyoscyamine, imipramine, itraconazole, , Levothyroxine, atorvastatin, lovastatin, meclizine, megesterol, mercaptopurine, metolazone, mometasone, , Nabumetasone, omeprazole, paroxetine, propafenone, quinapril, simvastatin, sirolimus, tacrolimus, tacrolimus, Tizanidine, fluvar Tatin (fluvastatin), pitavastatin (pitavastatin), pravastatin (pravastatin), rosuvastatin (rosuvastatin), or may comprise a mixture thereof.

As an example, the drug may be paclitaxel.

"Liposomes" of the present invention refer to vesicles comprising at least one bilayer separating the internal aqueous phase from the external aqueous environment.

In the present invention, the liposome is used as a method for solubilizing water-insoluble water-soluble drugs and soluble in alcohol, and nanoparticles containing albumin-bound drugs are sealed in the liposome to provide a drug delivery composition have.

In one example, the liposome may comprise phospholipids.

The "phospholipid" may be any one or more of neutral lipid, anionic lipid, and cationic lipid. The phospholipid is not limited as long as it is used in the art to produce a liposome for carrying a drug, and can be separated and purified from a plant, oil or the like, or recombinantly available commercially. For example, the neutral lipid may be selected from the group consisting of La-phosphatidylcholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphocholine , DOPC), 1,2-distearoyl-sn-glycero-3-phophocholine (DSPC), 1,2-dipalmitoyl-sn Glycero-3-phophocholine (DPPC), 1,2-diolauryl-sn-glycero-3-phosphoethanolamine (1,2-dipalmitoyl- -dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), 1,2-distearoyl-sn-glycero-3-phophoethanolamine, DSPE 2-dipalmitoyl-sn-glycero-3-phophoethanolamine (DPPE), cholesterol, isopropyl myristoyl myristate, or mixtures thereof.

The anionic lipids include L-alpha-phosphatidic acid, L-alpha-phosphatidyl-DL-glycerol, cardiolipin, La- L-aphosphatidylinositol, L-alpha-phosphatidylserine, 1,2-dilauryl-sn-glycero-3- [phospho-rac- (1-glycerol) (1,2-dilauroyl-sn-glycero-3- [phospho-rac- (1-glycerol)] DLPG, 1,2-dilauryl- Serine] (1,2-dilauroyl-sn-glycero-3- [phospho-L-serine], DLPS), 1,2- dilauroyl- glycero-3- [phospho-rac- (1-glycerol)] 1,2-dimyristoyl-sn-glycero-3 - [phospho-rac- (1-glycerol)], DMPG), 1,2-dimyristoyl-sn-glycero-3- phospho-L- Phosphino-L-serine, DMPS, 1,2-dimyristoyl-sn-glycero-3-phosphate, DMPA, Glycero-3- [phospho-rac- (1-glycerol)], 1,2-dioleoyl-sn-glycero-3- [phospho-rac- , DOPG), 1,2-dioleoyl-sn-glycero-3- [phospho-L-serine], DOPS) , 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-dipalmitoyl-sn-glycero- Glycero-3-phospho-L-serine, DPPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate (1 , 2-dipalmitoyl-sn-glycero-3-phosphate, DPPA), 1,2-distearoyl-sn-glycero-3- [phospho-rac- phospho-rac- (1-glycerol)], DSPG), 1,2-distearoyl-sn-glycero-3- [phospho-L- -sn-glycero-3- [phospho-L-serine], DSPS, 1,2-distearoyl-sn-glycero-3-phosphate, DSPA ), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy (polyethylene Glycero-3-phosphoethanolamine-N- [carboxy (polyethylene glycol) 2000], 1,2-distearoyl-sn- Glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) 2000]), 1,2-distearoyl-sn-glycerol 3-phosphoethanolamine-N- [PDP (polyethylene glycol) 2000] (1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- (1-palmitoyl-2-oleoyl-sn-glycero-3- [phospho-rac- (1-glycerol)] - , POPG), 1-palmitoyl-2-oleyl-sn-glycero-3- phospho-L-serine (1-palmitoyl-2-oleoyl-sn-glycero-3- , POPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, POPA, oleic acid, Lt; / RTI >

The cationic lipid is selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioleoyl glutamide, distearoyl- But are not limited to, distearoyl glutamide, dipalmitoyl glutamide, dioleoyl aspartamide, 1,2-dioleoyl-propane, 3-dimethylammonium-propane, DODAP), ß- [N- (N ', N'-dimethylaminoethanecarbamoyl] -Chol), dimethyldioctadecylammonium bromide (DDAB), or a mixture thereof.

Phospholipids such as POPC (1-Palmitoy-2-oleoyl- sn -glycero-3-phosphocholine), cholesterol, dioleoyl-1,2-diacyl- -distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene-glycol) -2000].

The molar ratio of POPC, cholesterol, and DOTAP as the phospholipid may be 4: 10: 1 to 5: 0.1 to 2.5, for example 6: 3: 1. The ratio of POPC and cholesterol is similar to that of cell membrane, and DOTAP can maximize the interaction with the albumin particle or cell membrane to be filled by providing positive charge to the phospholipid membrane and liposome formed as cationic lipids. Also, the DSPE-PEG2000 can be from 1 to 5% of the total phospholipid volume, for example 3%. This can minimize the adsorption of plasma proteins to pre-formed nanoparticles and provide stability during blood circulation.

In the present invention, water-insoluble water-soluble and alcohol-soluble drugs can be solubilized by preparing an alcoholic aqueous solution in the inner phase of the liposome and controlling the permeability to alcohol so that the alcohol contained therein is not liberated through the liposome membrane Liposomes can be constructed using low phospholipids.

Since the solubility of the phospholipids is particularly low in alcohol, the liposomes have high stability against the alcoholic aqueous solution constituting the inner phase.

Also, by enclosing the drug in a liposome, the biomolecular permeability of the encapsulated drug is enhanced by the interaction between the phospholipid bilayer of the liposome and the lipid membrane component of the biomembrane, and the biomembrane permeation enhancing effect is further enhanced by the alcohol contained in the liposome .

The drug delivery composition of the present invention may have a diameter of 50 nm to 250 nm. In another example, it may have a diameter of 150 nm to 210 nm. In addition, the drug delivery composition exhibits a uniform particle size distribution. Nanoparticles can pass easily through the blood vessels (EPR effect) in tissues such as cancer, inflammation, etc., which have very weak and loose structures because of their characteristic particle size. Thus, particles having diameters in this range are preferred for systemic drug delivery.

In another aspect, the present invention provides a method for preparing a drug delivery composition comprising a liposome encapsulated with nanoparticles containing an albumin-bound drug, the method comprising mixing an aqueous solution in which albumin is dissolved and an alcohol in which the drug is dissolved to form an alcoholic buffer solution And a second step of enclosing the nanoparticles prepared in the first step into the liposome, wherein the nanoparticles comprise a drug bound to albumin. And a method for producing the drug delivery composition.

Firstly, the first step may be to produce nanoparticles comprising a drug bound to albumin, dispersed in an alcohol using a desolvation technique.

Specifically, albumin is dissolved in distilled water, filtered through a filter, and then the pH is adjusted using a base. Drugs are also dissolved in ethanol. Thereafter, a solution containing the drug is added dropwise to the albumin solution to prepare nanoparticles containing the albumin-bound drug.

In addition, the first step may include ultrasonication to disperse the albumin-dissolving alcohol in a ratio of 1:10 to 1: 100, followed by dispersion in nanoparticle size .

In one example, the molar ratio of albumin and water-miscible drug in the mixture may be 1:50.

The alcohol may be a C _{1 -4} lower alcohol. In one embodiment of the present invention, ethanol may be used, but the present invention is not limited thereto.

The aqueous solution may be, for example, distilled water, but is not limited thereto.

The drug may be a water-soluble and alcohol-soluble drug, and may be bound to albumin and encapsulated in a liposome for delivery. Examples thereof are as described above.

In the second step, for example, a phospholipid is mixed and dissolved in an organic solvent (step A); (Step B); and a step of mixing and stirring the alcoholic buffer solution in which nanoparticles are dissolved, which comprises the phospholipid thin film prepared in step B and a drug bound to the albumin, to form a liposome (step C ).

Further, after the step C, the step of extruding the liposome through the extruder may be further included. The extruder may comprise, for example, a polycarbonate membrane.

The extrusion may be performed by passing a membrane having pores having a specific size under pressure so that a desired size of the liposome can be prepared by controlling the pore size of the polycarbonate, the pressure at the time of passage, the number of times of passage, and the like.

Specifically, as the pore size of the polycarbonate membrane is smaller, the pressure at the time of passage increases, and the number of times of passage increases, the size of the finally obtained liposome particles decreases.

In one embodiment of the present invention, an Avanti mini extruder was extruded through a stack of two polycarbonate membranes having a pore size of 200 nm to produce liposomes of uniform size ranging from 150 to 210 nm. The number of times of the extrusion is not particularly limited, and may be, for example, 1 to 50 times, and may be 11 times as an example of the present invention.

The composition for drug delivery comprising the liposome encapsulated with the nanoparticles containing the novel albumin-bound drug of the present invention is a drug delivery composition having uniform hydrodynamic size, high packing efficiency and loading capacity Can be provided. The delivery is characterized by a higher colloidal stability compared to the simple complex of paclitaxel and albumin similar to Abraxane ( ^R ). In addition, since the composition for drug delivery exhibits better in vitro and in vivo behavior such as better release profile, cell uptake and cytotoxicity than paclitaxel-albumin simple complex and paclitaxel dissolved in crepolm / ethanol, the paclitaxel- May be usefully employed as a means for the delivery of soluble drugs.

1B is a TEM image of (a) empty liposome, (b) APNs and (c) L-APNs, Albumin, (C) APNs, (D) L-APNs, and (E) empty solid liposome formulations are shown in (d) FT-IR, (e) DSC thermogram and (f) XRD. Figure ID shows the SEC results for different formulations ((A) pure PTX, (B) pure albumin, (C) APNs, (D) L-APNs and (E) empty liposomal formulations).
Figure 2A shows DLS, Figure 2B shows the colloidal stability after dilution of APNs (?) And L-APNs (?) Determined by SEC. (Blue: undiluted; red: 10-fold dilution; and green: 100-fold dilution). Figure 2c shows the stability of L-APNs or serum () after storage at 25 ^° C (○) and 4 ^° C (●).
3A shows the in vitro release profile of PTXs of APNs and L-APNs in PBS buffer (pH 7.4, 0.14 M NaCl) at 37 ° C, FIG. 3b shows Higuchi plots of APNs and L- . The data was expressed as mean SD (n = 3).
FIG. 4A shows the results of incubation for 24 hours and 48 hours, followed by B16F10, FIG. 4B shows the results of the measurement of nanoparticles (black), pure PTX (red), APNs (light blue?), And L- APNs (dark blue ▼) in vitro cytotoxicity.
FIG. 5A shows cell cycle analysis after treatment of MCF-7 cells with 0.1 μg / mL of pure PTX, APNs and L-APNs, respectively, and 1.0 μg / mL of FIG. 5B after 6 hours and 24 hours of culture.
FIG. 6 shows in vitro cell uptake of various formulations into B16F10 and MCF-7 cells, FIG. 6A shows nanoparticles (green), APNs (pink) and FACS analysis of L-APNs (blue); FIG. 6b shows CLSM analysis of cells (Hoechst dye: blue, AlexaFluor® 488-albumin: green and rhodamine-phospholipid: red) after treatment with drug-free nanoparticles, APNs and L-APNs; Figure 6c shows a schematic cell uptake measurement by LC-MS / MS.
FIG. 7A shows plasma concentration-time profiles after intravenous administration of pure PTX, APNs and L-APNS to mice; FIG. 7B shows the biodistribution and tumor accumulation profile of pure PTX, APNs and L-APNs in tumor mice 2 hours after intravenous administration. The recorded values are expressed as mean SD (n = 4). * P <0.0001 compared to APN, and P <0.0001 compared to PTX.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

<Materials Preparation and Statistical Analysis>

Paclitaxel (PTX) was purchased from TCL Co., LTD (Tokyo, Japan). Bovine serum albumin (MW = 66,000) and 3- (4,5-dimethylthiazol-2-yl) -2,5-dimethylthiazolium bromide (3- -2-yl) -2,5-diphenyl tetrazolium bromide, MTT) was obtained from Sigma-Aldrich (Yongin, South Korea) as 1-palmitoyl-2-oleoyl-sn- 2-oleoyl- sn -glycero-3-phosphocholine, POPC), dioleoyl-1,2-diacyl-3-trimethylammonium propane (dioleoly- trimethyl ammonium propane, DOTAP), 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N- [methoxy (polyethylene- glycero-3-phosphoethanolamine-N- [methoxy (polyethylene-glycol) -2000], DSPE-PEG2000) and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL, USA).

The results of the following experimental examples are shown as mean ± SD (standard deviation) for four separate measurements. ANOVA or t-test was used to determine whether the differences between the following experimental groups were statistically significant and considered to be statistically significant when p <0.05.

Production Example 1  : Albumin Combined  Nanoparticles containing drugs ( APN )

APN was prepared using the desolvation technique. Specifically, bovine serum albumin (BSA) was dissolved in distilled water at a concentration of 1 mg / ml, filtered through a 0.45 μm filter, and the pH was adjusted to 9 using 0.2 M NaOH. Separately, PTX was dissolved in 100% ethanol at a concentration of 13 mg / ml. 10 占 퐇 of the PTX solution prepared above was added dropwise to 200 占 퐇 of the albumin solution and mixed with stirring. Thereafter, the mixed solution was subjected to sonication for 1.5 hours in order to uniformly disperse the nanoparticles. As a result, the final formulation containing 1% of ethanol, i.e., albumin-bound paclitaxel nanoparticles (APNs) was obtained.

As a result of measuring the particle size of the APN formulation, it was found that the particle size was uniform, and the average particle size was ~ 105 nm. This was slightly smaller than that of the abrasive nanoparticles (Abraxane (130 nm) and Doxil ^® (150 nm), which are nanoparticles containing the same drug.

Manufacturing example  2: minimum size ANP Manufacturing

The APN formulation prepared by the method of Preparation Example 1 at a molar ratio of BSA: PTX (200: 130 μg / mL concentration) = 3: 150 showed the minimum particle size. At this molar ratio, it was observed that approximately PTX 6 molecules bound to site 1 of the BSA subdomains IIA and IIIA. As a result, the particle size was increased ten times as compared with BSA nanoparticles (~ 10 nm).

Production Example 3  : APN end Enclosed Of liposomes (L-APNs)  Produce

Lipids of POPC (6 μmol), cholesterol (3.0 μmol), DOTAP (1 μmol) and DSPE-PEG2000 (0.3 μmol) were dissolved in chloroform (CHCl ₃ ) and then chloroform was removed using a rotatory evaporator A cationic lipid thin film was prepared. Thereafter, 1 ml of an APN solution (1 ml) dispersed in 1% ethanol prepared in Preparation Example 1 was added to the dried cationic lipid thin film, and the mixture was incubated with intermittent mixing at room temperature for 4 hours. As a result, 10 mM of lipid was finally obtained. The lipid-dispersed dispersion was extruded 11 times with two polycarbonate membrane stacks each having a pore size of 200 nm using an extruder (Avanti mini extruder) to prepare uniform size liposomes.

Example  One: Fluidic  size, Polydispersity index ( PDI ), &lt; RTI ID = 0.0 &gt; Morphology  Measure

The hydrodynamic size, polydispersity (PDI) and zeta-potential of the L-APN formulation of Preparation Example 3 were measured And analyzed using dynamic light scattering (DLS). The L-APN formulation was appropriately diluted with distilled water and measured at 25 캜 using a particle size analyzer (ELS-Z (Photal, Osaka, Japan)). The morphologies of the different formulations were measured using a transmission electron microscope (TEM; H7600, Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV. The results of the measurement are shown in Fig. 1B ((a) is empty liposome, (b) is APN and (c) is L-APN).

The particles of L-APN of Preparation Example 3 have a uniform size distribution and have an average size of 180 to 210 nm (PDI, to 0.090) larger than empty liposomes (Fig. 1 (c)).

In addition, the zeta potential of the empty liposome shows a strong positive charge of about 24 mV, whereas the zeta potential of the L-APN formulation shows a significantly smaller charge of 4 mV or less (Table 1). This difference appears because the positive charge of the empty liposome is partially compensated by the negative charge of the APN, indicating that the APN is well encapsulated in the liposome.

Particle size (nm) PDI ζ-Potential (mV) Encapsulation Efficiency (%) Loading Capacity (%) APNs 106.2 ± 3.12 0.101 + 0.02 -2.65 ± 0.986 _ _ Blank liposome 179.5 ± 2.13 0.086 ± 0.01 23.96 + 0.381 _ _ L-APNs 209.7 ± 4.17 0.090 + 0.03 3.69 ± 1.265 99.96 ± 0.01 6.5 ± 0.10

The data are expressed as mean + - SD (n = 3).

TEM measurements can be used to confirm the size of the L-APN in the dry state as well as the structural morphology in the three-dimensional space after coloring (FIG. 1B). In particular, for all detected L-APNs, the particles were characterized by distinct monodisperse spherical morphologies.

In particular, the L-APN exhibits a sharp morphological morphology (Fig. IB (c)), whereas the APN is clearly stained (Fig. 1b (b)). In FIG. 1B, a residue mass formed after the evaporation of the particle clusters or the solvent was also observed.

On the other hand, although the shape observed by TEM is the same as the DLS measurement, the size by TEM appears smaller than that by DLS. This difference may be due to the fact that the DLS measurement reflects the particle size of the hydrodynamics in its swellon state, while the TEM measures the diameter in the dried state.

Example  2: Physical properties of nanoparticle formulation

(One) FT - IR  analysis

FT-IR spectra were recorded using a Thermo Scientific Nicolet Nexus 670 FT-IR Spectrometer and Smart iTR software with a diamond window (Thermo Fisher Scientific Inc., Waltham, Mass.). Spectra with a resolution of 32 times per sample, 4 cm ^-1, was recorded by scanning the range of 4000 cm ^-1 at 550.

The FT-IR spectrum is used to measure chemical interactions between the drug and protein / liposome functional groups, and the spectra of pure PTX, APN and L-APN are shown in Figure Ic (d). Pure PTX is ^{2965 cm -1 (= CH),} 1707 cm -1 (C = O group), 1641 cm -1 (CC stretch), 1370 cm -1 (CH 3 bending, 1250 cm ^-1 (CN stretch), 1072 cm ^-1 (CO stretch), and 709 cm ^-1 (CH off the plane). Also, since these peaks are present and not significantly changed in the spectrum of liposome formulations, it was confirmed that there is no chemical interaction between PTX and proteins or liposomes.

(2) differential scanning calorie ( DSC ) analysis

Differential scanning calorimeter (DSC-Q200, TA Instruments, New Castle, DE, USA) was used to study the thermal properties of the samples. And the temperature was elevated at a temperature raising rate of 10 ° C / min within the range of 40 ° C to 250 ° C by the differential scanning calorimeter (DSC).

Figure 1c (e) shows DSC thermograms of pure PTX, APNs and L-APNS. The pure PTX showed a sharp endothermic peak at 224 ° C. The disappearance of this transition in both the APN and L-APN formulations indicates that the PTX is in a more amorphous molecular state. The hydrophobic interactions of PTX and BSA, which are important for obtaining maximum entrapment, are believed to help stabilize the amorphous form and promote encapsulation in the vesicle structure.

(3) X-ray diffraction  analysis

The X-ray diffraction (XRD) pattern of the sample was recorded using a vertical goniometer and an X-ray diffractometer (X'Pert PRO MPD diffractometer, Almelo, The Netherlands).

1C (f) shows XRD patterns of pure PTX, APNs and L-APNs. The XRD patterns of pure PTX show many sharp and strong peaks at scattering angles (2?) Of 10.81, 11.92, 12.90, 15.26, 16.81, 21.56, 25.089 and 42.16 °, indicating a high degree of crystallinity. In contrast, the diffraction pattern of the APN and L-APN formulations did not show such a peak indicating that the drug was present in a generally amorphous form. As a result, it was found that APN and L-APN had higher crystallinity than PTX.

Example  3: Drug loading ( loading ) And measurement of the sealing efficiency

The drug loading efficiency was measured by ultrafiltration using a centrifugal filter (Amicon centrifugal filter device, molecular weight cut off [MWCO], 10000 Da, Millipore, Billerica, MA, USA) followed by HPLC. 20 占 퐇 of the filtered sample was injected into a C18 column (Sepax BR-C18, 5 占 퐉 120 占 4.6 占 150 mm). The mobile phase was a mixture of acetonitrile and water in a ratio of 1: 1, and a flow rate of 1.0 ml / min was used.

To detect unencapsulated PTX, 500 [mu] l of the liposomal formulation was filtered through centrifugal ultrafiltration at 14,000 rpm for 30 minutes. The content of PTX in the filtrate was measured using HPLC with UV monitoring at 227 nm. The drug loading (loadin) and encapsulation efficiency were calculated using the following equation.

It was confirmed by size exclusion chromatography (SEC) that APN was contained in the liposome of L-APN formulation at 90% or more (FIG. 1d). Specifically, pure albumin was eluted at a retention time of 4.417 min with an intensity of 500, and the empty liposome was eluted at a retention time of 2.703 at an intensity of 600. L-APNs were eluted as two distinct peaks, one with a retention time of 2.703 minutes at 1200 MHz and had the same retention time as the empty liposomes, confirming that the APN formulation was encapsulated in liposomes And the other exhibits an unencapsulated APN having a retention time equal to that of pure albumin, eluted at a retention time of 4.417 minutes at 150 cycles. As a result of analyzing the peak intensity of L-APN, it was confirmed that more than 90% of APN was encapsulated in the liposome.

As a result, it was confirmed that the filling efficiency and loading ability for L-APN formulations were ~ 99% and ~ 6.5%, respectively.

Example  4: In vitro drug release profile analysis

Release of PTX from each formulation prepared in Preparation Examples 2 and 3 was measured by dialysis. Specifically, 1 ml of each formulation was placed in a dialysis bag (MWCO, 3500; Membra-Cel ^® , Viskase, USA) and soaked in 10 ml of phosphate-buffered saline containing 0.1% Tween 80 (PBS, pH 7.4). To measure drug release, 0.2 ml of medium was collected at various time points and the new medium was replaced by the same volume.

The concentration of PTX in each sample was determined by LC-MS / MS using an Agilent HPLC (1260 series, USA) coupled with a 6490 Triple quadrupole mass spectrometer equipped with an ESI Agilent jet stream ion source. Docetaxel was used as an internal standard and the following MS ionization parameters were used: positive ESI mode, Argon collision gas, capillary voltage of 5 kV; Gas temperature of 225 ° C, gas flow of 15.1 I / min, impact energy for paclitaxel of 30 eV, impact energy for docetaxel of 22 eV and source temperature of 40 ° C.

The assay was quantified using multiple reaction monitoring (MRM) by observing ion changes of paclitaxel 876.3-308.0 m / z and docetaxel 830.3-303.9 m / z .

The in vitro release profiles of APN and L-APN are shown in Figures 3a and b.

As shown in Figure 3A showing the drug release profile of each formulation, the APN released a biphasic release profile, i.e. 20% of the total PTX within 12 hours from the beginning and 26% up to 72 hours And release profiles of up to 75% over the next 7 days. This feature is due to the initial release phase of the drug present on the nanoparticle surface.

On the other hand, L-APN showed a consistent release over the measurement period: 6% of PTX within the first 12 hours, 10% at 24 hours, and 30% after 7 days. This slow release rate is believed to be due to the longer path length of the PTX in the support. That is, PTX has a longer passage length because it must first be separated from the hydrophobic core of albumin and dispersed through the liposome bilayer.

This release profile can be beneficial to the patient in that it effectively increases the solubility of the drug and protects the drug and has a significantly extended release profile at physiological pH values. Thus, the occurrence of the EPR phenomenon is enabled. In addition, prolonged serum half-life can increase the likelihood of spraying into the tissue between the tumors.

To analyze the profile for emissive dynamics, data for both formulations was applied to the Higuchi's model. The plotted PTX release profile for the square root of the time during the first 24 hours showed a smaller slope versus L-APN than the release profile of APN as control (Figure 3b).

The data was also applied to the Korsmeyer-Peppas relation. Where n represents the emission mechanism. The n value for L-APN based on this data was 0.65 (Table 2). This corresponds to non-Fickian kinetics and indicates that one or more mechanisms, such as dispersion and corrosion, are associated with PTX release.

Model Higuchi R ² Korsemeyer - Peppas
Model R ² n
APNs 0.9395 0.9249 0.987 L-APNS 0.9931  0.9293 0.694

R ² : Crystal constant; n: dispersion or release index (n = 3)

Example  5: L- APN Study of colloidal stability of formulations

The colloidal stability of the formulation was measured by dynamic light scattering (DLS) in a refrigerator (4 ° C) or at room temperature (25 ° C) for 1 month. Dilution-induced degradation was measured using a size-exclusion column (Zenic SEC-300, 3 μm 300 A, 4.6 × 150 mm) chromatography at different dilutions.

Specifically, storage and colloidal stability of the formulations were measured after storage at low temperature (4 ° C) or room temperature (25 ° C) for one month and the colloid stability of the formulations was studied in fetal bovine serum (FBS). For each 20 μl of empty albumin, empty liposome, APN or L-APN at various dilutions (× 0, × 10 and × 100), a size-exclusion column (size -exclusion column, Zenic SEC-300, 3 μm 300 A, 4.6 × 150 mm) and observed with a UV spectrometer at 215 nm.

The particle size of the ANP formulation began to increase immediately upon dilution with water and increased up to 20-fold at the highest dilution (Fig. 2a). On the other hand, the particle size of the L-APN formulation remained constant in dilution. Consistently, L-APN was readily detectable by SEC at higher dilutions. On the other hand, ANP was not detected at the corresponding dilution (Fig. 2B). As a result, it was found that the particle size of the L-APN formulation did not increase unlike the APN formulation even when the degree of dilution was increased. As a result, the colloid stability was confirmed.

In addition, it was confirmed that L-APN was very stable for 1 month in PBS or serum media in long-term storage at 4 and 25 ° C (FIG. 2C).

Example  6: Study of cell uptake

The cellular uptake of the formulations was analyzed qualitatively and quantitatively using confocal laser scanning microscopy (CLSM), flow cytometry and LC-MS / MS in B16F10 and MCF-7 cells.

(One) CLSM Of Cellular Absorption by Cells

Cells (2 × 10 ³ ) from each cell line were inoculated into a 35-mm dish and cultured at 37 ° C. for 24 hours to improve cell adhesion. The cells were then incubated with different formulations at a concentration of 200 μg / ml for 1 hour and washed three times with cold PBS. Cells were fixed with PBS for 4 minutes at room temperature with 4% paraformaldehyde, and then the cells were washed twice with PBS again. The nuclei were stained with 1 ml of 1 μg / ml Hoechst dye for 2 min and washed with PBS. The coverslips containing the cells were then placed on the slides and observed using a confocal laser microscope (CLSM).

(2) In a flow cytometer  Quantitative analysis of cellular uptake by

Cells (3 x 10 ⁵ ) from each cell line were inoculated into a 35-mm dish and grown to 90% under tissue culture conditions. The cells were incubated with APN and L-APN formulations at 37 ° C for 1 hour and then washed twice with PBS to remove unbound nanomaterials. Cells were separated using 0.25% trypsin / EDTA, centrifuged twice at 1500 rpm for 3 minutes, and redispersed in 500 [mu] l PBS. Dispersed cells were cultured in BD FACS Calibur ^TM (USA) Into a flow cytometer and analyzed for excitation and emission wavelengths of 495 and 625 nm, respectively, to detect rhodamine. Untreated cells were used as a control and analyzed at least 10,000 times per sample to obtain results.

(3) LC - MS / MS Quantitative Analysis of Cellular Absorption by

Inoculated with B16F10 and MCF-7 cells in 6-well plates at a density of 2 × 10 ⁵ / well of cells, and the cells were cultured for 24 hours. Cells were treated with ANP and L-APN formulations for 0.5, 1 and 3 h. The cells were then washed twice with PBS, trypsinized, and centrifuged (10,000 rpm x 10 min, 4 &lt; 0 &gt; C). The granules were sonicated in an ice bath for 30 seconds at 200 W for 10 seconds duration at 10 sec intervals by 30 cycles of Bioruptor ultrasonic treatment. The supernatant was then collected and extracted for determination of drug concentration using LC-MS / MS.

Cellular uptake of empty liposomes, APN and L-APN into B16F10 and MCF7 cells was measured qualitatively by flow cytometry (FACS) and confocal scanning laser microscopy (CLSM) using Alexa Fluor 488 As a result, as shown in FIG. 6A, the FACS analysis confirmed that the fluorescence intensity of all cell types increased after treatment for all formulations, and the fluorescence intensity indicates absorption of the nanoparticles. In particular, the absorption of L-APN was significantly higher than that of empty liposomes and APNs in all cell lines. In addition, higher cell uptake of L-APNs was confirmed by CLSM. Initially, L-APN was found to accumulate in the cytoplasm after being dispersed into surrounding regions containing nuclei. At equilibrium, many of the green and red fluorescence are observed in the cytoplasmic region (Figure 6b).

Based on these observations, it was found that the nanoparticles were initially absorbed through the endocytosis to the endosomal region, followed by degradation of the nanoparticles and dispersion through the cytoplasm. Where the drug is thought to act on the microtubule.

In addition, the cellular uptake of PTX to B16F10 and MCF-7 cells after treatment with empty liposomes, APN and L-APN was quantitatively determined with LC-MS / MS over time. Cellular uptake of PTX increases in proportion to time after treatment with APN or L-APN in all lines (Figure 6c). Higher uptake was observed in B16F10 cells. The maximum PTX concentration measured after L-APN treatment was 12.55 and 18.28 ng / ml for MCF-7 and B16F10 cells, respectively. Additionally, L-APN showed significantly higher uptake than APN for all cell lines at all time points measured. Similar to FACS analysis, LC-MS / MS analysis confirmed the time dependence of intracellular PTX concentration after L-APN treatment. The PTX concentration increased immediately for 2 hours after treatment but decreased thereafter. In particular, the PTX concentration in MCF-7 cells was 3-fold higher after 1 hour than 30 minutes. However, it remained relatively constant until 2 hours later. This is due to the initial flux internalization of L-APN. That is, subsequent cell uptake does not increase since the cell has already internalized the formulation. Similar trends were also observed in B16F10 cells. That is, there was almost no increase until 120 minutes after a double increase within 60 minutes.

As a result, it was confirmed that the L-APN formulation showed significantly higher cell uptake than the APN formulation.

Example  7: In vitro  Cytotoxicity analysis

Cytotoxicity of pure PTX, empty liposomes, APN, L-APN was measured using MTT assay. Cells were exposed to each formulation, incubated for 24 and 48 hours, and treated with MTT solution. The absorption rate was measured at a wavelength of 570 nm using a microplate reader.

Specifically, mouse melanoma cells B16F10 (1 x 10 ⁴ ) or breast cancer cells MCF-7 were inoculated into 96-well plates and cultured at 37 ° C for 24 hours. The cells were then treated with pure PTX, empty liposomes, APN and L-APN and incubated at 37 ° C for 24 hours and 48 hours. The cells were washed twice with PBS and maintained in DMEM medium (Dulbecco ' s modified Eagle ' s medium) containing 10% FBS and 1% P / S. Ten microliters of a 5 mg / ml solution of MTT was added to each well and the sample was incubated at 37 占 for 2 hours in the dark. Cells were separated and formazan crystals were dissolved in 100 μl of DMSO and absorbance was measured at a wavelength of 570 nm using a microplate reader.

As shown in Figs. 4A and 4B, only slight cytotoxicity was observed after 48 hours for any of the above formulations. Indicating that the carrier is fully biocompatible and suitable for in vivo evaluation. Both APN and L-APN exhibit measurable cytotoxicity dependent on dose and time. Both APN and L-APN showed cytotoxicity in a concentration range (0.001-10 μg / mL) corresponding to the expected serum levels in humans. The results show that L-APN shows cytotoxicity to the extent that it corresponds to pure PTX.

Example  8: Cell cycle analysis

The DNA content of the cells was measured using flow cytometry and the percentage of cells present at each cell cycle stage was measured using flow cytometry software (BD FACS Comp ^TM software).

MCF-7 cells inoculated into 6-well plates were treated with pure PTX, APNs and L-APN (PTX concentration 0.1 μM or 1 μM) at 37 ° C for 6 to 24 hours. After the cells reached a density of approximately 1 x 10 ⁶ / well, the cells were harvested by trip myth, centrifuged at 3000 rpm for 3 minutes at 4 ° C, and washed twice with ice-cold PBS. Fixed with 70% cold ethanol, and incubated on ice for 1 hour. The cells were treated with 5 μl of a 10 mg / ml ribonuclease solution and stained with 1 μg / ml propidium iodide solution at 37 ° C. for 30 minutes in the dark state. The DNA content of the cells was measured using a flow cytometer and the ratio of the remaining cells in each cell cycle was measured using flow cytometry software (BD FACS Comp ^TM software).

In addition, the cytotoxic effects of pure PTX, APN and L-APN were measured by cell cycle analysis after culturing in MCF-7 cells for 6 or 24 hours at a concentration of PTX 0.1 or 1 μg / ml, 5a and 5b.

PTX binds to a [beta] -tubulin dimer in microtubules and induces G ₂ / M arrest of rapidly differentiating cells, leading to apoptosis. As shown in FIG. 5, the proportion of cells in the G ₂ / M phase was increased for PTX nanoparticle formulations. This indicates that cell death is intensified.

Interestingly, L-APN induces a higher rate as compared to other dosage forms than a higher rate of sub--G ₁ cells, i.e., 3.27% and 2.18% of ANP and pure PTX to 12.29% after 6 hours. After 24 hours exposure to each formulation (L-APN, APN and PTX), the percentage of killed cells increased to 35%, 7.75% and 6.78%, respectively. This indicates that the enhanced cellular apoptosis observed for the L-APN formulation affects the toxicity of the PTX component of the liposome. At any rate, this enhancement of PTX activity may be a very favorable improvement for clinical patients.

Example  9: Pharmacokinetics, biological distribution and tumor accumulation

To measure pharmacokinetic and tumor accumulation parameters in vivo, male BALB / c mice and C57BL6 tumor mice (20-25 g), each 4 weeks old, were used. Twelve mice were divided into three groups administered intravenously with 4 doses of pure PTX, APN or L-APN at a dose of 5 mg / kg (100 μL of a 1 mg / mL solution). Blood and organ samples were collected, processed, and analyzed by LC-MS / MS. Pharmacokinetic profiles for pure drugs and formulations were applied to non-compartmental models using WinNonLin software (v 4.0, Pharsight Software, Mountain View, Calif.).

Specifically, all experimental protocols related to animals were approved by the Gachon University Animal Ethics Committee (Gachon University, South Korea). To measure pharmacokinetic parameters in vivo, 4-week-old 20-25 g male BANB / c mice were used. Twelve mice were separated into three experimental groups of four mice per group. Each group received 5 mg / kg of pure PTX, APNs, or L-APNs intravenously (100 μL of 1 mg / mL solution). The pure drug was dispersed in PEG400. 10 μl of blood was collected from the right saphenous vein into heparinized tubes (100 IU / ml) from each mouse at time points of 3, 10, 20 or 30 minutes and 1, 2, 4 and 8 hours after administration, RTI ID = 0.0 &gt; 14,000 rpm. &Lt; / RTI &gt; The supernatant serum layer was collected and stored at -20 ° C.

(1) Serum sample process

The frozen serum samples were thawed at room temperature, and then 9 μl of a 1: 1 mixture of acetonitrile and 0.1% formic acid in 1: 1 and 1 μl of docetaxel were added. The PTX was then extracted and mixed with 0.25 ml ethyl acetate for 3 minutes to precipitate the protein. The sample was centrifuged at 10,000 rpm for 10 min and the organic layer was evaporated in vacuo for 30 min and redispersed in 20 μl of mobile phase (0.1% formic acid and acetonitrile (1: 1 v / v) MS analysis. Chromatography was performed under the same conditions as described in the in vitro release study.

(2) Tumor accumulation

C57BL6 mice bearing B16F10 tumors were randomly divided into three groups (four animals) and treated with pure PTX, APN or L-APN (100 μL of a 1 mg / mL solution) at a dose of 5 mg / kg PTX by intravenous injection Respectively. Two hours after the injection, PBS containing heparin at a constant rate was sprayed through an anesthetized mouse via a cardiac puncture to obtain a pressure of 25 mm Hg. The tumors and target organs (heart, lung, liver and kidney) were then removed and immediately washed with PBS to remove any adherent material. The organs were collected in pre-weighed EP tubing and analyzed for PTX content. 1 ml of PBS was added to the pre-weighed organs and sonicated in an ice bath for 15 seconds at 15 W intervals at 200 W by 30 cycles of Bioruptor ultrasonic treatment. The prepared homogenous suspension proceeded to an extraction process for preparing the above-mentioned samples, and the quantitative analysis of PTX was carried out in LC-MS / MS as mentioned in the release study section.

(3) Pharmacokinetic data analysis

Pharmacokinetic profiles for pure drugs and formulations were applied to non-compartmental models using WinNonLin software (v 4.0, Pharsight Software, Mountain View, Calif.). The time area under the serum concentration vs. time curve from 0 to infinity (AUC _{0 -∞),} elimination half-life (t _1/2), and clearance (clearance, Cl) was determined from the data.

The Cl value was calculated as dose / AUC _{0 -8h} and the mean residence time (MRT) was calculated as the sum of the central and tissue portions.

After intravenous administration of each formulation, the serum concentration-time profile of PTX was measured. As a result, after the initial abrupt release, the serum concentration of PTX rapidly decreased with pure PTX or APN, and then 2 ng / lt; RTI ID = 0.0 &gt; ml &lt; / RTI &gt; (Figure 7a). This was confirmed by the initial dispersed phase of two or more phases. PTX clearance is similar to pure PTX and APN. These results are consistent with the in vitro colloidal stability analysis of APN showing rapid degradation in serum. In particular, after administration of a single dose of L-APN, PTX generally remains in the bloodstream at a higher concentration than with APN over time. The pharmacokinetic parameters measured by non-compartmental analysis are shown in Table 2 above. The AUC _{0 -∞} for L-APN was 668.01 ng / l · h, which is four times higher than the AUC _{0 -∞} for pure PTX or APN ( p <0.0001).

In addition, L-APN has a clearance of 3 to 4 times lower than that of pure PTX or APN, 1.5 to 2 times greater retention time, longer serum half-life and lower removal rate constant.

In vivo tissue distribution of PTX was also measured in B16F10 tumor mice (5 mg / kg PTX) after intravenous administration of pure PTX, APN and L-APN. PTX was rapidly dispersed in all major organs after intravenous administration. The highest concentration of drug was observed in the liver, followed by kidney and lung. In particular, L-APN was preferentially accumulated in tumors as compared to pure PTX and APN (FIG. 7B).

Claims (14)

A composition for drug delivery, comprising a liposome encapsulated with a nanoparticle containing an albumin-bound drug, wherein the drug is paclitaxel.
The method according to claim 1,
Wherein the liposome comprises a phospholipid.
3. The method of claim 2,
The phospholipids are selected from the group consisting of POPC (1-Palmitoy-2-oleoyl- sn -glycero-3-phosphocholine), cholesterol, DOTAP (Dioleoyl-1,2-diacyl- -sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene-glycol) -2000]).
4. The composition for drug delivery according to claim 3, wherein the volume ratio of POPC, cholesterol, DOTAP and DSPE-PEG2000 is 4 to 10: 1 to 5: 0.1 to 2.5: 0.1 to 0.5.
A first step of preparing nanoparticles dispersed in an alcoholic buffer solution by mixing an aqueous solution in which albumin is dissolved and an alcohol in which paclitaxel is dissolved; And
And a second step of enclosing the nanoparticles prepared in the first step into the liposome,
The method of claim 1, wherein the nanoparticle comprises paclitaxel bound to albumin.
The method for preparing a drug delivery composition according to claim 5, wherein the alcohol is a C _1-4 lower alcohol.
6. The method of claim 5, wherein the molar ratio of albumin to paclitaxel is 1: 10-100.
6. The method of claim 5, wherein the second step comprises:
Mixing the phospholipids and dissolving them in an organic solvent (step A);
Drying the phospholipid dissolved in the organic solvent to form a thin phospholipid (step B); and
And a step (C) of forming a liposome by mixing and stirring the phospholipid thin film prepared in the step B with the alcoholic buffer solution in which the nanoparticles prepared in the first step are dispersed Way.
9. The method of claim 8,
Wherein the phospholipid is at least one selected from the group consisting of POPC, cholesterol, DOTAP and DSPE-PEG2000.
The method of claim 8, wherein the organic solvent is at least one selected from the group consisting of chloroform, ether, acetone, alcohol, and carbon disulfide.
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