Functionalized liposomes useful for the delivery of bioactive compounds

Abstract

The invention relates to conjugates in which a sterol is functionalized by an ether bond with a water-soluble polymer to which a guiding ligand is bound. These conjugates improve the physico-chemical and delivery properties of their carrying vesicles, making these more stable, homogeneous and effective. A method for their preparation, a pharmaceutical composition containing said liposomes, and their therapeutic use are described as well.

Claims

1. A conjugate, comprising: i) a sterol; ii) a chain of polyethylene glycol having a proximal end and a distal end wherein said chain of polyethylene glycol is covalently bound by its proximal end to i) via an alkyl ether bond; and iii) a guiding ligand, capable of selectively binding to one or several receptors present in a target cell, said guiding ligand being covalently bound to the distal end of ii); wherein the guiding ligand is a peptide.

2. The conjugate of claim 1 wherein the sterol is cholesterol.

3. The conjugate of claim 1 wherein the chain of polyethylene glycol has a number of repetitions from 2 to 10.

4. The conjugate of claim 1 wherein the guiding ligand comprises an RGD sequence.

5. The conjugate of claim 4 wherein the guiding ligand is the peptide of sequence SEQ ID NO: 1.

6. A liposome comprising the conjugate of claim 1.

7. The liposome of claim 6, having a monomodal particle size distribution.

8. The liposome of claim 7 wherein the average particle size is from 25 up to 500 nanometers, and the average Z potential in absolute value is from 20 up to 90 mV.

9. The liposome of claim 6, further comprising a therapeutic agent.

10. The liposome of claim 9 wherein the therapeutic agent is α-galactosidase.

11. A method of delivering a therapeutic agent to a subject comprising administering the liposome of claim 9 to the subject.

12. A method of treatment comprising administering a therapeutically effective amount of the liposome of claim 9 together with pharmaceutically acceptable excipients or carriers, to a subject having Fabry disease.

13. A pharmaceutical composition comprising a therapeutically effective amount of the liposome of claim 9, together with pharmaceutically acceptable excipients and/or carriers.

14. A method for the preparation of the conjugate of claim 1 comprising the following steps: a) reacting a sterol with a sulfonyl halide in the presence of a base and a solvent to give the corresponding sulfonyl ester; b) reacting the compound obtained in the step a) with a polyethylene glycol with a number of repetitions from 2 to 10 in the presence of a solvent; c) activating the compound obtained in the step b) with disuccinimidyl carbonate; and d) reacting the compound resulting from the step c) with a peptide comprising the RGD sequence in the presence of a base and a solvent.

15. A method for the preparation of the liposome of claim 6 comprising the following steps: a) preparing an aqueous solution which may optionally include a surfactant; b) preparing a solution comprising: I) a conjugate, the conjugate comprising: i) a sterol; ii) a chain of polyethylene glycol having a proximal end and a distal end wherein said chain of polyethylene glycol is covalently bound by its proximal end to i) via an alkyl ether bond; and iii) a guiding ligand, capable of selectively binding to one or several receptors present in a target cell, said guiding ligand being covalently bound to the distal end of ii); and II) cholesterol and, optionally, a phospholipid dissolved in an organic solvent, where the organic solution is expanded with a compressed fluid; c) optionally, adding a therapeutic agent either to the solution of step a), or to the solution of the step b) before expanding this solution; and d) depressurizing the solution resulting from step b) over the resulting solution of step a).

16. A liposome comprising the conjugate of claim 4.

17. The liposome of claim 16, further comprising a therapeutic agent.

18. The liposome of claim 16, wherein the therapeutic agent is α-galactosidase.

19. A method of treatment comprising administering therapeutically effective amount of the liposome of claim 17, together with pharmaceutically acceptable excipients or carriers, to a subject having Fabry disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Assembly illustrating the method for the preparation of vesicles of the invention (Abbreviations: R: High-pressure vessel; V: Valve; P: Pump; F: Flow-meter; H: Heat-exchanger; ST: Stirrer; FL: Filter; TI: Temperature Indicator; PIC: Pressure indicator and Controller; C: Collector; RD: Rupture Disc)

(2) FIG. 2. Particle-size distribution of vesicles containing conjugates of the invention (with ether bond, in dashed line) and vesicles containing conjugates with carbamate bond (in solid line). In the figure it is shown that the former have a monomodal distribution, while the latter show a bimodal, less homogeneous distribution.

(3) FIG. 3. Confocal images showing the results of the internalization experiments for liposomes bearing no conjugate, bearing DPPC:Col:Col-PEG-RGD(ether) and bearing DPPC:Col:Col-PEG-RGD(carbamate).

(4) Left-hand side, middle and right-hand side columns correspond to Transmission (Cells), Green Channel (Lysosomes) and Red Channel (DiD) respectively. Upper, middle and lower rows correspond to DPPC:Col:Col-PEG-RGD(ether), DPPC:Col:Col-PEG-RGD(carbamate) and DPPC:Col respectively.

(5) FIG. 4. Shows the fluorescence intensity associated to the cells that have internalized the DID-labelled liposomes. It is shown that this internalisation is significantly higher when using DPPC:Col:Col-PEG-RGD(ether) liposomes than when using DPPC:Col:Col-PEG-RGD(carbamate) and DPPC:Col liposomes.

(6) The y axis shows Fluorescence Intensity (a.u.). In the x axis: the left-hand side value, middle value and right-hand side value correspond to plain liposomes, liposomes with the DPPC:Col:Col-PEG-RGD(ether) conjugate and liposomes with the DPPC:Col:Col-PEG-RGD(carbamate) conjugate respectively.

(7) FIG. 5. Alpha galactosidase activity of liposomes in MAEC cultures of alpha galactosidase-deficient mice, at a concentration of 1.5 μg/ml of enzyme. The y axis depicts % Gb3 loss (mean+/−standard deviation). In the x axis: point 1 corresponds to alpha-galactosidase (no liposome encapsulation), point 2 corresponds to functionalized liposomes (either with carbamate or ether linked conjugates) and point 3 corresponds to the functionalized liposomes which have been diafiltered.

(8) FIG. 6. Effect of the diafiltered conjugate-bearing liposomes carrying alpha-galactosidase at different concentrations on the loss of Gb3 in alpha-galactosidase deficient endothelial cells. The x axis shows % Gb3 loss whereas the y axis shows the alpha-galactosidase concentration (μg/mL). The line with the solid black dots corresponds to liposomes bearing conjugates with the carbamate bond, whereas the line with the empty squares corresponds to liposomes bearing conjugates with the ether bond.

EXAMPLES

Example 1. Preparation of Conjugates

(9) Conjugates which form part of the invention (with ether bond between the cholesterol and the PEG), and those which have been described in the state of the art (with carbamate bond between the cholesterol and the PEG) were synthesized with the steps detailed below. Acronyms used in the experimental summary are:

(10) ACN: Acetonitrile.

(11) CDI: 1,1′-Carbonyldiimidazole

(12) DCM: Dichloromethane

(13) DIPEA: N,N-Diisopropylethylamine

(14) DMF: N,N-Dimethylformamide

(15) DSC: Disuccinimidyl carbonate

(16) HPLC-MS: High Performance Liquid Chromatography-Mass Spectrometry

(17) HPLC-PDA: High Performance Liquid Chromatography-Mass Spectrometry-Photodiode Array Detector

(18) NMR: Nuclear Magnetic Resonance

(19) TMBE: Methyl tert-butyl ether

(20) TFA: Trifluoroacetic acid

(21) TIS: Triisopropylsilane

Example 1a: Preparation of Conjugates with Ether Bond

Preparation of Cholesterol Tosyl (A)

(22) ##STR00001##

(23) Into a flask, the cholesterol (1 eq., 1.006 g) was introduced, dissolved in anhydrous pyridine (12 mL). Tosyl chloride (2 eq., 1.004 g) was then added and the solution was left under stirring for 24 h at room temperature. Over the solution were added 5 mL of water and the crude was extracted with DCM (3×6 mL). Et.sub.2O (total solution) was added in the organic phase and then dried over MgSO.sub.4, filtered and the solvent was removed to dryness. The crude was recrystallized from petroleum ether, obtaining a white solid (0.969 g, 70%).

(24) HPLC-PDA: (C18, 5-100% B, A: ACN B: MeOH, 4.5 min, 2 mL/min, λ=210 nm) r.sub.T: 2.7 min (89%) .sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 0.65 (s, 3H); 0.85 (d, 1.6 Hz, 3H); 0.87 (d, 2 Hz, 3H); 0.90 (d, 6.8 Hz, 3H); 0.96 (s, 3H); 2.44 (s, 3H); 5.3 (m, 1H); 7.33 (d, 8.0 Hz, 2H); 7.79 (d, 8.3 Hz, 2H). .sup.13C NMR: (100 MHz, CDCl.sub.3) δ: 144.52 (C32), 139.00 (C4), 134.85 (C35), 129.87 (C34, C36), 127.77 (C33, C37), 123.65 (C7), 82.54 (C2), 56.79 (C11), 56.25 (C17), 50.05 (C10), 42.43 (C12), 39.80 (C22), 39.65 (C13), 39.01 (C3), 37.03 (C5), 36.49 (C20), 36.31 (C18), 35.89 (C6), 31.99 (C9), 31.89 (C8), 28.77 (C1), 28.33 (16), 28.15 (C15), 24.38 (C23), 23.95 (C21), 22.95 (C25), 22.70 (C24), 21.77 (C38), 21.13 (C14), 19.28 (C26), 18.84 (C19), 11.98 (C27).

Preparation of Cholesterol-Tetraethylene Glycol (B)

(25) ##STR00002##

(26) Into a flask, 0.503 g of cholesterol tosyl (A) was added and dissolved in 5 mL of anhydrous 1,4-dioxane. 3.591 g of tetraethylene glycol (20 eq.) were added on this solution and the mixture was reacted for 4 h at reflux under argon atmosphere. The resulting solution was concentrated to dryness and then dissolved in 20 mL of DCM and washed with 2×20 mL sat. NaHCO.sub.3, 3×20 mL H.sub.2O, 1×20 mL sat. NaCl. The resulting aqueous phases were extracted again with 20 mL DCM. Finally, after combining the organic phases were dried over MgSO.sub.4, filtered and the solvent was evaporated to dryness. The purification of crude was carried out by chromatography with basic alumina (DCM/MeOH 0-5%). 0.260 g of product as a yellowish oil were obtained (50%).

(27) HPLC-PDA: (C.sub.18, 5-100% B, A: ACN B: MeOH, 4.5 min, 2 mL/min, λ=210 nm) r.sub.T: 2.6 min (73% 210 nm). .sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 0.67 (s, 3H); 0.85 (d, 1.6 Hz, 3H); 0.87 (d, 1.6 Hz, 3H); 0.91 (d, 6.4 Hz, 3H); 0.99 (s, 3H); 3.18 (m, 1H); 3.67 (m, 16H). .sup.13C NMR: (100 MHz, CDCl.sub.3) δ: 141.07 (C4), 121.71 (C7), 79.69 (C2), 72.82-70.40 (C30, C32, C33, C35, C36, C38), 67.36 (C29), 61.87 (C39), 56.93 (C11), 56.30 (C17), 50.33 (C10), 42.47 (C12), 39.93 (C22), 39.66 (C13), 39.11 (C3), 37.37 (C5), 37.01 (C6), 36.33 (C20), 35.92 (C18), 32.09 (C8), 32.04 (C9), 28.43 (C1), 28.37 (C16), 28.15 (C15), 24.43 (C23), 23.97 (C21), 22.96 (C24), 22.70 (C25), 21.21 (C14), 19.51 (C26), 18.86 (C19), 12.00 (C27).

Preparation of Cholesterol-Tetraethylene Glycol-DSC (C)

(28) ##STR00003##

(29) The compound B (1 eq., 0.2746 g) was dissolved in 9 mL of a mixture of DCM:ACN:DIPEA (1:1:1) and added DSC (3 eq., 0.2746 g). The mixture was stirred under argon atmosphere and after 16 h it was noted the disappearance of the starting product B. The solvent was evaporated to dryness and the crude was dissolved in 5 mL DCM. This organic phase was washed with 5 mL of water. The organic phase was dried over MgSO.sub.4, filtered, and finally, the solvent was evaporated to dryness. The crude was used in the following reaction without any further purification.

Preparation of cRGDfK (D)

(30) ##STR00004##

(31) The synthesis of D-peptide was carried out as described in Dai X., et. al. “An improved synthesis of a selective αvβ3-integrin antagonist cyclo (-RGDfK)” Tetrahedron Letters 2000, vol. 41, pp. 6295-6298, with minor modifications. Briefly, the final cyclic peptide was deprotected with a mixture of TFA/TIS/H.sub.2O (95:2.5:2.5) and purified by RP-HPLC, obtaining the D-peptide (180.9 mg, 15%) as a white solid.

(32) HPLC-MS: (C.sub.18, 5-100% B, A: ACN B: NH.sub.4HCO.sub.3 20 mM, 3.5 min, 1.6 mL/min, λ=210 nm) r.sub.T: 2.47 min, m/z=604.3 [M+H].sup.+, Calculated mass: 603.67 (99% 210 nm). MALDI-TOF (ACH): 604.24 [M+H].sup.+. ANALYSIS OF AMINO ACIDS: Asp: 0.673, Gly: 0.734, Arg: 0.780, Lys: 0.677, Phe: 0.632

Preparation of Cholesterol-cRGDfK (E)

(33) ##STR00005##

(34) Compound C (1.5 eq., 69.00 mg) was dissolved in 5 mL of anhydrous DMF and DIPEA (2 eq., 32 μL) and D-peptide (1 eq., 65.95 mg) were added. The reaction was left under stirring for 16 h until the disappearance of the D-peptide (control by HPLC-MS). The solvent was removed and the crude was precipitated from TMBE (3×). The compound E was obtained as a white solid (55.5 mg, 74%). HPLC-MS: (Symmetry300 C.sub.4, 5-100% B, A: ACN B: H.sub.2O, 30 min, 1 mL/min, λ=210 nm) r.sub.T: 17.31 min, m/z=1192.8 [M+H].sup.+, Calculated mass: 1192.53. HPLC-PDA: (C.sub.4, 5-100% B, A: ACN B: H.sub.2O, 30 min, 1 mL/min, λ=210 nm) r.sub.T: 20.1 min (98% 210 nm). ANALYSIS OF AMINO ACIDS: Asp: 0.99, Gly: 1.18, Arg: 1.15, Lys: 0.92, Phe: 1.04

Example 1b (Comparative with Example 1a): Preparation of Conjugates with Carbamate Bond

Preparation of Cholesterol-DCl (F)

(35) ##STR00006##

(36) The cholesterol (1 eq., 200.2 mg) was dissolved in 9 mL of a mixture of DCM/DIPEA/ACN (1:1:1) and to this the CDI (10 eq., 852.1 mg) was added. It was allowed to react overnight, and the appearance of the desired product was observed. The solvent was evaporated to dryness and the crude was dissolved in 5 mL DCM. This organic phase was washed with 5 mL of water. The organic phase was dried over MgSO.sub.4, filtered and finally the solvent was evaporated to dryness. The crude was used in the following reaction without any further purification.

Preparation of Cholesterol-Tetraethylene Glycol Carbamate (G)

(37) ##STR00007##

(38) The compound F (1 eq., 109.6 mg) was taken and dissolved in DCM, to this solution was added 1-amino-3,6,9-trioxaundecanyl-11-ol (4.5 eq., 152.8 mg) and the solution was then set to pH 8 using DIPEA. The mixture was allowed to react, monitoring its progress by HPLC-MS, until it was observed the disappearance of the starting product F. The crude was washed making extractions from the organic phase with water. The organic phase was dried over MgSO.sub.4, filtered and finally the solvent was evaporated to dryness. The final crude was purified by silica column (Isocratic AcOEt) to obtain 80.2 mg of compound G as a yellowish oil (60%). HPLC-MS: (XSelect C.sub.18, 5-100% B, A: ACN B: MeOH, 4.5 min, 2 mL/min, λ=210 nm) r.sub.T: 1.87 min m/z=606.47 [M+H].sup.+, Calculated mass: 605.89. HPLC-PDA: (C.sub.4, 5-100% B, A: ACN B: H.sub.2O, 30 min, 1 mL/min, λ=210 nm) r.sub.T: 21.8 min (95% 210 nm) .sup.1H NMR: (400 MHz, CDCl.sub.3) δ: 0.680 (s, 3H); 0.86 (d, 1.8 Hz, 3H); 0.87 (d, 1.8 Hz, 3H); 0.91 (d, 6.5 Hz, 3H); 1.01 (s, 3H); 3.36 (m, 1H); 3.65 (m, 16H). 13C NMR: (100 MHz, CDCl.sub.3) δ: 156.58 (C29), 140.07 (C4), 122.53 (C3), 70.74 (C8), 70.56-70-25 (C33, C35, C36, C38, C39, C41), 61.79 (C42), 56.83 (C14), 56.27 (C15), 50.15 (C6), 42.45 (C13), 40.85 (C32), 39.88 (C22), 39.66 (C12), 38.75 (C7), 37.15 (C5), 36.71 (C10), 36.32 (C20), 35.93 (C18), 32.05 (C1), 32.02 (C2), 28.37 (9), 28.35 (C17), 28.15 (C16), 24.43 (C23), 23.97 (C21), 22.96 (C24), 22.70 (C25), 21.18 (C11), 19.48 (C19), 18.85 (C27), 12.00 (C26).

Preparation of Cholesterol-Tetraethylene Glycol-DSC Carbamate (H)

(39) ##STR00008##

(40) The compound G (1 eq., 80.2 mg) was taken and reacted with DSC (10 eq., 316.8 mg) in 9 mL of a DCM/DIPEA/ACN mixture (1:1:1) for 16 h. When the disappearance of the compound G was detected, the solvent was evaporated to dryness and the crude was dissolved in 5 mL of DCM and washed with water. The organic phase was dried over MgSO.sub.4, filtered and the solvent was evaporated, obtaining a crude ready to use without any further purification.

Preparation of Cholesterol-cRGDfK Carbamate (I)

(41) ##STR00009##

(42) The entire compound H was dissolved in 5 mL of anhydrous DMF and DIPEA (2 eq., 60 μl) and D-peptide (1 eq., 51.2 mg) were added. The reaction was allowed under stirring for 16 h until the disappearance of the D-peptide (control by HPLC-MS). The solvent was removed and the crude was precipitated from TMBE (3×), finally washed with water. In this way 17 mg of the product I were obtained as a white solid.

(43) HPLC-MS: (XSelect C.sub.18, 5-100% B, A: ACN B: MeOH, 4.5 min, 2 mL/min, λ=210 nm) r.sub.T: 1.03 min, m/z=1235.94 [M+H].sup.+, Calculated mass: 1235.55. HPLC-PDA: (C.sub.4, 5-100% B, A: ACN B: H.sub.2O, 30 min, 1 mL/min, λ=210 nm) r.sub.T: 19.91 min (86% 210 nm). ANALYSIS OF AMINO ACIDS: Asp: ND, Gly: 1.23, Arg: 1.11, Lys: ND, Phe: 1.11.

Example 2. General Method for the Preparation of Vesicles

(44) The vesicles carrying the conjugates of the invention (ether bond) or the conjugates of the state of the art (carbamate bond) were prepared as is described below, based on a procedure described elsewhere (Cano-Sarabia, M. et. al. “Preparation of Uniform Rich Cholesterol Unilamellar Nanovesicles Using CO2-Expanded Solvents” Langmuir 2008, vol. 24, pp. 2433-2437).

(45) The method for the preparation of vesicles was carried out in an assembly such as that represented in FIG. 1. The assembly consisted of a high pressure reactor (R) to which was added a solution with the components of the vesicle membrane in ethanol at a certain concentration (C1, C2 . . . Cn depending on the number of components), at atmospheric pressure and at the working temperature (T.sub.w=T). In a second step, compressed CO.sub.2 was added up to the working pressure (P.sub.w=P), yielding the volumetric expansion of the solution to a molar ratio X.sub.CO2. The addition was carried out using the pump P1 through the valve V-1, keeping closed the rest of the valves. The system was maintained at a pressure P and temperature T for a certain amount of time to ensure the total homogenization and the thermal equilibrium. After this time, V-4 was opened with the purpose of connecting the reactor R with the filter FL, previously pressurized with N.sub.2 up to P.sub.w, keeping closed the rest of the valves. The opening of V-6 allowed the depressurization of the volumetrically expanded solution on an aqueous solution pumped through P2. In this final step, a stream of N.sub.2 added through V-2 to P.sub.w was used as a plunger to push the expanded solution, and to maintain the working pressure constant in the reactor during the step of depressurization. The presence of the filter FL allowed to collect any precipitates formed during the process. The vesicles formed were collected in the vessel C, and then stored in glass bottles at 4° C. Once the depressurization was completed, V-6 and V-2 were closed and depressurization of the equipment by opening again V-6 was carried out.

Example 3. Preparation of Vesicles of DPPC:Cholesterol:Cholesterol-PEG-RGD by the Compressed Fluid Technology

(46) First a solution of 8 mg of Cholesterol, 24 mg of DPPC and 4 mg of conjugate (cholesterol-PEG-RGD) in 1.2 mL of ethanol was introduced in a high pressure reactor with a volume of 7.5 mL, at atmospheric pressure and working temperature (T.sub.w=35 C). Compressed CO.sub.2 was added, yielding the volumetric expansion of the solution to a molar ratio X.sub.CO2=0.8 and a working pressure P.sub.W=10 MPa. To achieve the total homogenization and thermal equilibrium, the system was left for approximately 60 minutes at 10 MPa and 35° C. Finally, the expanded organic solution was depressurized from the working pressure to the atmospheric pressure, over 24 mL of an aqueous solution. In this last step, a stream of N.sub.2 at 10 MPa was used as a plunger to push the cholesterol solution in ethanol in order to maintain the working pressure constant in the reactor during the depressurization. The vesicles were then transferred to a vessel which, when sealed, was stored at 5±3° C. until use.

(47) As a result, vesicles of DPPC:Cholesterol:cholesterol-PEG-RGD (10:6:1) were obtained with a microscopic appearance, average size and Z-potential shown in the Table 1.

(48) The average size, particle-size distribution and Z-potential were determined by DLS (Dinamic Light Scattering) at a temperature of 25 degrees Celsius.

(49) Table 1 shows the results of physical appearance, average particle size and Z-potential of different batches of vesicles DPPC:cholesterol:cholesterol-PEG-RGD.

(50) TABLE-US-00001 TABLE 1 Average size.sup.3 (nm) Z-Potential.sup.3 Composition (±SD).sup.4 PDI.sup.3 (±SD).sup.4 (mV) (±SD).sup.4 DPPC:Chol:Chol-PEG- 152.7 (±0.8) 0.182 (±0.010) +32.4 (±2.4) RGDether.sup.1 (1.3 mM:0.8 mM:0.1 mM) 128.8 (±0.2) 0.235 (±0.017) +30.4 (±1.4) DPPC:Chol:Chol-PEG- 141.5 (±1.8) 0.357 (±0.005)  −9.5 (±0.2) RGDcarb.sup.2 (1.3 mM:0.8 mM:0.1 mM) 117.9 (±0.6) 0.357 (±0.008)  −0.9 (±0.3) .sup.1Vesicles of DPPC:Cholesterol:Cholesterol-PEG-RGD with ether bond, .sup.2Vesicles of DPPC:Cholesterol:Cholesterol-PEG-RGD with carbamate bond, .sup.3Measurements of DLS carried out by an instrument Nano-ZS (Malvern Instruments, United Kingdom). PDI: Polydispersity index .sup.4SD: Standard deviation of three consecutive measurements on the same batch.

(51) In regard to the macroscopic appearance of different formulations, it was observed that both the vesicles prepared using Cholesterol-PEG-RGD with ether bond and the vesicules prepared using Cholesterol-PEG-RGD with carbamate bond had the appearance of an opalescent, dispersed solution. On the other hand, it was noted that in the case of vesicles of DPPC:Cholesterol:Cholesterol-PEG-RGD with ether bond the different preparations of vesicles had no stability problems in the short term, with small average size and polydispersity index, which make them attractive from the pharmaceutical point of view. Such polydispersity index is higher in the case of vesicles Cholesterol-PEG-RGD with carbamate bond, the distribution of which is bimodal, in contrast to the monomodality found for vesicles containing Cholesterol-PEG-RGD with ether bond (FIG. 2). For this latter system higher Z-potentials were found (around +30 mV), falling within the value range considered to allow the colloidal stability of dispersed systems over time. It was therefore concluded that in terms of both particle size and suspension stability, vesicles having conjugates with the ether bond have better properties than the vesicles having conjugates with the carbamate bond.

Example 4. Internalization Experiments

(52) In order to assess whether the liposomes carrying the conjugates of the invention have superior properties in terms of intracellular delivery of their contents in comparison with liposomes carrying conjugates of the prior art, an experiment of internalization of the contents of the liposomes in a cell line was carried out. The substance delivered was a dye that can be easily monitored by fluorescence.

(53) Liposome Labelling

(54) For liposome labelling, 500 μL of plain liposomes (DPPC:Col liposomes) were directly mixed with 25 μL of a Did ethanolic solution (1 μM) for a final concentration of 50 nm of the dye in the membrane. After 30 min of mixing, free DiD was separated from the total sample by gel filtration. For this purpose pre-packed columns (PD SpinTrap G-25) were three times equilibrated with PBS buffer and then 130 μL of sample was added. The separation took place by spin centrifugation at 800×g.

(55) The procedure described above was repeated analogously for the other liposome types.

(56) Cell Culture.

(57) CDC/EU.HMEC-1 (HMEC-1) cells were provided by Centers for Disease Control and Prevention (CDC-NIDR). HMEC-1 is an immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells. HMEC-1 cells were maintained in MCDB 131 (Invitrogen) supplemented with 50 units ml-1 penicillin, 50 μg ml-1 streptomycin, 10 mM L-glutamine and 10% fetal bovine serum (FBS) in a 37° C. humidified atmosphere with 5% CO2. All the media, serum and antibiotics were purchased from Invitrogen.

(58) Cellular Uptake of Liposomes Assessed by Laser Scanning Confocal Microscopy (LSCM).

(59) HMEC-1 cells were seeded onto Fluorodish culture plates (World Precision Instruments, Sarasota, Fla.) at a density of 2×105 cells per plate and allowed to grow for 36-48 hours. 50 μl of DiD-labelled Liposomes (DPPC:Col) or DiD-labelled Liposome-RGD conjugates (DPPC:Col:Col-PEG-RGDether liposomes and DPPC:Col:Col-PEG-RGDcarbamate liposomes) (1.5 mg/ml) were mixed with 200 μl MCDB 131 medium, added into the cells and incubated for 3 h at 37° C. in a humidified atmosphere with 5% CO2. Subsequently, cells were washed with serum-free MCDB 131 and incubated at 37° C. for 5 min with Lysotracker Green DND-26 (50 nM, Molecular probes, Eugene, Oreg.) to label the endosomal/lysosomal compartments. Cells were examined under an inverted Leica SP5 laser scanning confocal spectral microscope (Leica Microsystems Heidelberg GmbH, Mannheim, Germany) using a 60×1.42 NA oil immersion objective. To visualize two colours of fluorescence simultaneously, we used the 514 nm line from Argon laser for Lysotracker green and the 630 nm line from a He—Ne laser for Did.

(60) Confocal Images for DPPC:Col:Col-PEG-RGDether liposomes (top), DPPC:Col:Col-PEG-RGDcarbamate liposomes (middle) and DPPC:Col (bottom) liposomes are shown in FIG. 3. The spots observed in the red channel (right-hand side column) show the presence of labelled liposomes internalized by the cells. It is clearly observed that for DPPC:Col:Col-PEG-RGDether liposomes this internalization is higher in comparison with the other two.

(61) Flow Cytometry.

(62) HMEC-1 cells were seeded at densities of 2×105 cells ml-1 on Fluorodish culture plates (World Precision Instruments, Sarasota, Fla.) 36-48 h prior to experiment. Cells were incubated with DiD-labelled Liposomes (DPPC:Col) or DiD-labelled Liposome-RGD conjugates (DPPC:Col:Col-PEG-RGDether liposomes and DPPC:Col:Col-PEG-RGDcarbamate liposomes) (0.3 mg/ml) resuspended into MCDB 131 supplemented with 10 mM L-Glutamine without FBS for 3 hours at 37° C. Cells were subsequently washed twice with Dulbecco's phosphate buffered saline (DPBS) solution, detached using trypsin and resuspended in cell culturing medium before subjecting to fluorescence-activated cell sorting analysis. Data acquisition and analysis was performed using FACS scan (Beckton-Dickinson) and BD FACSDiva software. 10.000 viable cells were evaluated in each experiment.

(63) In FIG. 4 it can be seen the fluorescence intensity associated to the cells that have internalized the DID-labelled liposomes. It is shown that this internalisation is significantly higher when using DPPC:Col:Col-PEG-RGDether bearing liposomes than when using DPPC:Col:Col-PEG-RGDcarbamate bearing liposomes or simple DPPC:Col liposomes bearing no conjugate.

Example 5. In Vitro Activity Experiments

(64) Activity Assays:

(65) Primary cultures of mouse aortic endothelial cells (MAEC) of GLA (alpha galactosidase) deficient mice (GlatmKul1) were isolated following procedures previously described (Shu L., et al. “An in vitro model of Fabry disease” J. Am. Soc. Nephrol. 2005, vol. 16, pp. 2636-45). Endothelial origin of isolated cells was confirmed by CD105 staining.

(66) For activity assays, cells in passages 2 to 5 were seeded in 24 well plates and maintained at 37° C. and 5% of CO2. Twenty-four hours after seeding 8 μM of NBD-Gb3 (Matreya) was added to the cultures along with the specified concentrations of tested compounds (free enzyme—alpha-galactosidase, enzyme containing liposomes or empty liposomes). After 48 h incubation, cells were trypsinized and Gb3-NBD fluorescent signal was analyzed by flow cytometry (FacsCalibur, Beckton Dickinson). To calculate the percentage of Gb3-NBD signal, fluorescent signal in control cells (without treatment) was established as 100% and the rest of the values were normalized accordingly. Since alpha-galactosidase activity reduces those Gb3 deposits, the percentage of Gb3 loss (% Gb3 loss=100−% Gb3-NBD signal) was used to plot the results.

(67) It can be seen in FIG. 5 the alpha galactosidase activity of liposomes in MAEC cultures of alpha galactosidase-deficient mice. 1.5 μg/mL of free enzyme (alpha-galactosidase-histogram bar number 1 in FIG. 5) reduced the Gb3 deposits in 92.32%. Liposomes incorporating the same enzyme reduced the Gb3 deposits similarly (88.9%) independently of having carbamate or ether linkage (histogram bars number 2 in FIG. 5). Purification of liposomes by diafiltration reduced slightly this activity (histogram bars number 3 in FIG. 5), however, it is worth noting that the concentration of the enzyme in these purified liposomes will be lower than 1.5 μg/mL, since just a fraction (usually around 80%) of the total enzyme used in the liposome preparation is finally encapsulated.

(68) It is also shown in FIG. 6 that the diafiltered liposomes carrying the conjugates with the ether bond have a higher activity than those carrying the conjugates with the carbamate bond at varying concentrations.

Example 6. Improvement of Specific Enzymatic Activity Due to the Encapsulation of Alpha-Galactosidase in the Liposomes Bearing the Conjugates of the Invention (with an Ether Bond)

(69) Production of Recombinant GLA (Alpha-Galactosidase)

(70) The expression vector pOpinE-GLA encodes a full-length version of the human α-galactosidase (GLA) gene, cloned into pOPINE plasmid (Berrow N S, et al. “A versatile ligation-independent cloning method suitable for high-throughput expression screening applications” Nucleic Acids Res 2007, vol. 35, e45). The suspension-adapted HEK (human embryonic kidney) cell line FreeStyle™ 293 F (Gibco, Invitrogen corporation) was used to produce recombinant GLA by means of PEI-mediated transient gene expression. Details of GLA production and purification have been previously described (Corchero J L, et al., “Integrated approach to produce a recombinant, his-tagged human alpha-galactosidase a in mammalian cells”, Biotechnol Prog. 2011, vol. 27, pp. 1206-1217).

(71) Detection and Quantification of Recombinant GLA Encapsulated into Liposomes

(72) To estimate the incorporation of recombinant GLA into liposomes, samples from 1) initial GLA in water, 2) GLA mixed with lipids, 3) purified liposomes and 4) water containing free, non-encapsulated GLA, were mixed with denaturing, loading buffer and analyzed by SDS-PAGE and further western-blot developed with a rabbit polyclonal anti-GLA serum from Santa Cruz Biotechnology (a-gal A H-104: sc-25823) and a goat anti-rabbit IgG HRP-conjugate (Bio-Rad Laboratories, Inc., cat. #170-6515) as secondary antibody. Amounts of recombinant GLA within each of the above mentioned samples were estimated by comparison with known amounts (usually ranging from 25 to 125 ng) of a recombinant GLA previously produced, purified, and quantified in our laboratory. Samples to be quantitatively compared were run in the same gel and processed as a set. Densitometric analyses of the bands were performed with the Quantity One software (Bio-Rad Laboratories, Inc). Percentage of encapsulated GLA was obtained by comparison of amounts of GLA found in purified liposome fractions with total, initial amount of GLA added.

(73) Characterization of Recombinant GLA Encapsulated into Liposomes

(74) The enzymatic α-galactosidase activity of GLA found into the different samples was assayed, in vitro, fluorometrically as described by Desnick et al. (Desnick R J, et al. “Fabry's disease: enzymatic diagnosis of hemizygotes and heterozygotes. Alpha-galactosidase activities in plasma, serum, urine, and leukocytes”, J. Lab. Clin. Med. 1973, vol. 81, pp. 157-171) with the modifications of Mayes et al. (Mayes J S., et al. “Differential assay for lysosomal alpha-galactosidases in human tissues and its application to Fabry's disease” Clin. Chim. Acta 1981, vol. 112, pp. 247-251). Briefly, enzymatic activity was assayed using as substrate 4 methylumbelliferyl α D-galactoside (4MUG, Sigma Chemical), at a concentration of 2.46 mM in assay buffer (0.01 M acetic acid, pH 4.5). A typical assay reaction mixture contains 100 μl of substrate and 25 μl of enzyme sample. Enzymatic reactions took place in agitation, at 37° C. for 1 hour, and were stopped with 1.25 mL of 0.2 M glycine-NaOH buffer (pH 10.4). The released product (4-methylumbelliferone or 4-MU) was determined by fluorescence measurement at 365 and 450 nm as excitation and emission wavelengths, respectively. Samples containing from 0 to 500 ng 4-MU/ml of commercial 4-MU (Sigma Chemical) in 0.2 M glycine-NaOH buffer (pH 10.4) were used to create a standard curve to calibrate the readings. Specific enzymatic activities are expressed as μmol 4-MU/h/mg protein.

(75) Stability of Recombinant GLA into Liposomes.

(76) The stability of GLA into the different samples was assayed by following their specific enzymatic activity. After their preparation (“Day zero”), GLA protein amount and enzymatic activity were determined (as described before) for each sample. With those values, initial specific enzymatic activity was determined. Samples were kept in water at 4° C., and at different time points, enzymatic activity was assayed. Using initial amounts of GLA, specific enzymatic activities were recalculated and compared to that determined at “Day zero”, used as a reference.

(77) Efficiency of Encapsulation of GLA into the Liposomes.

(78) For each encapsulation experiment, the following samples were obtained and analyzed: 1. Initial GLA (before encapsulation). 2. Total GLA, that is, the result of adding the lipids to point 1 and encapsulating a fraction of the initial GLA. Total means both encapsulated and non-encapsulated GLA. The separation of GLA encapsulating liposomes from free GLA remaining in solution gives points 3 and 4. 3. Liposome-encapsulated-GLA, that is, only GLA which has been encapsulated. 4. Free GLA, that is, GLA which has not been encapsulated.

(79) The encapsulation efficiency of GLA into the vesicles, as determined by SDS-PAGE and further western-blot in different experiments, is shown in the next table:

(80) TABLE-US-00002 Efficiency of Experiment # Sample μg GLA/ml GLA encapsulation A ″Total″ (2) 3.87 SUVs-GLA (3) 0.63 16% Free GLA (4) 3.67 95% B ″Total″ (2) 4.17 SUVs-GLA (3) 1.23 29% Free GLA (4) 2.53 61% C ″Total″ (2) 4.31 SUVs-GLA (3) 0.97 23% Free GLA (4) 3.88 90% D ″Total″ (2) 7.90 SUVs-GLA (3) 2.32 29% Free GLA (4) 5.30 67% E ″Total″ (2) 7.70 SUVs-GLA (3) 2.27 29% Free GLA (4) 3.90 51% F ″Total″ (2) 6.40 SUVs-GLA (3) 2.28 36% Free GLA (4) 3.40 53%

(81) According to these results, the efficiency of GLA encapsulation into vesicles is of 27+/−6.8% (mean+/−standard deviation).

(82) TABLE-US-00003 Specific enzymatic avtivity Experiment # Sample μmol 4MU/h/mg GLA A Initial GLA (1) 312 Total (2) 1349 SUVs-GLA (3) 1570 B Initial GLA (1) 362 Total (2) 1112 SUVs-GLA (3) 957 C Initial GLA (1) 221 Total (2) 1239 SUVs-GLA (3) 1716 D Initial GLA (1) 109 Total (2) 1542 SUVs-GLA (3) 1750 E Initial GLA (1) 81 Total (2) 1454 SUVs-GLA (3) 1780 F Initial GLA (1) 109 Total (2) 2001 SUVs-GLA (3) 1934

(83) The addition to the GLA solution of lipids that will form the vesicles results in a significant increase in the specific enzymatic activity of the encapsulated enzyme.

(84) As can be seen, the specific enzymatic activity of “Total” samples (GLA mixed with the lipids that form the vesicles) clearly increases (from 4- to 18-fold) when compared to initial GLA, still not associated with vesicles or their components.

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