WO2012085888A2 - Polyelectrolyte complex, process for its manufacture and use thereof - Google Patents

Polyelectrolyte complex, process for its manufacture and use thereof Download PDF

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WO2012085888A2
WO2012085888A2 PCT/IB2011/055928 IB2011055928W WO2012085888A2 WO 2012085888 A2 WO2012085888 A2 WO 2012085888A2 IB 2011055928 W IB2011055928 W IB 2011055928W WO 2012085888 A2 WO2012085888 A2 WO 2012085888A2
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tmc
polyelectrolyte complex
pecs
chitosan
gal
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PCT/IB2011/055928
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French (fr)
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WO2012085888A3 (en
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Marina Inés GIANNOTTI
Fausto Sanz Carrasco
Simon Schwartz Navarro
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Centro De Investigación Biomédica En Red En Bioingeniería, Biomateriales Y Nanomedicina
Fundació Privada Institut De Bioenginyeria De Catalunya
Universitat De Barcelona
Fundació Institut De Recerca Hospital Universitari Vall D'hebron
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin

Definitions

  • the present invention relates to a carrier and delivery system for proteins, in particular a polyelectrolyte complex (PEC).
  • PEC polyelectrolyte complex
  • the present invention particularly relates to a polyelectrolyte complex designed to release the protein in lysosomes.
  • ERT enzyme replacement therapy
  • Fabry disease is a rare metabolic chromosome X-linked lysosomal storage disorder caused by the deficiency of a-galactosidase A (a-Gal A or a-GAL), the enzyme responsible for the degradation of globotriaocylceramide (Gb3 or GL-3) and other neutral glycosphingolipids.
  • a-Gal A or a-GAL a-galactosidase A
  • Gb3 or GL-3 globotriaocylceramide
  • Lysosomal storage leads to distension of the intracellular organelle and subsequent cellular dysfunction that translates into the presentation of renal, cardiac and cerebrovascular manifestations [2, 3].
  • ERT with exogenously administrated recombinant a-GAL has been shown to effectively address some of the clinical complications shown associated with Fabry disease [1, 3-5].
  • this approach is not completely effective since the enzyme is partly removed from the circulation by the liver and the spleen.
  • PECs polyelectrolyte complexes
  • nanoparticulate PEC species play an important role in the blood circulation, transport in cells and subsequent cellular routing [8-10].
  • An empirical hydrodynamic diameter for efficient cellular uptake and systematic delivery of nanoparticulate material has been established to be up to 200 nm [10, 1 1]).
  • the chosen polyelectrolyte should have optimal properties for self-assembly with the protein and effective release in the target intracellular compartment.
  • Chitosan nanoparticles have been used as carrier in protein and gene delivery for a number of different applications [6, 14-26]. Its outstanding characteristics such as non-toxicity, biocompatibility, biodegradability and low cost make it one of the most widely used biopolymers in biomedical applications [27, 28]. Chitosan is a natural aminopolysaccharide, the second-most abundant polysaccharide after cellulose.
  • Chitosan and its derivatives are cationic polymers (cationic polyelectrolytes) that can interact and form colloidal complexes with negatively charged biomacromolecules (DNA, proteins, polysaccharides) with nanoencapsulations purposes.
  • cationic polymers cationic polyelectrolytes
  • DNA negatively charged biomacromolecules
  • proteins proteins, polysaccharides
  • Quaternization of the amine groups of the chitosan molecule is one of the most explored strategies to overcome this drawback [30, 31].
  • TMC trimethyl chitosan
  • the present inventors have surprisingly found that by thoroughly tailoring the PECs components and their proportions, desirable properties (size, Zeta potential, colloidal stability and pH-dependence of their integrity) are achieved allowing the formation of stable vehicles without altering the protein functionality, as well as their internalization by cells and trafficking to the target organelles (lysosomes) via a pH- triggered mechanism.
  • the first object of the present invention is to provide a polyelectrolyte complex with the characteristics to be used as a delivery and carrier system.
  • said system is a carrier and delivery system for proteins and more particularly to be delivered in lysosomes in endothelial cells.
  • the second object of the present invention is to provide a process for manufacturing said polyelectrolyte complex according to the first object of the invention in a very simple way without using organic solvents or extreme conditions that may endanger the protein structure.
  • the third object of the present invention is to use said PECs for manufacturing a medicament useful for treating a lysosomal disease, in particular Fabry disease.
  • the present invention relates to a polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm.
  • the present invention also relates to a process for manufacturing said polyelectrolyte complex.
  • the present invention further relates to the use of said polyelectrolyte complex as a carrier and delivery sustem for proteins, in particular to be delivered to lysosomes and more particularly in endothelial cells.
  • the present invention additionally relates to use of said polyelectrolyte complex for the manufacture of a medicament for treating a lysosomal disease, in particular Fabry disease.
  • Figure 2 a) AFM image (height, tapping mode) of a TMC/cc-GAL PEC in a mass ratio of 0.82 with TPP (TMC/TPP mass ratio: 3.6), on an HOPG substrate in buffer HEPES 10 mM, pH 7.5; b) profile from the AFM topographic image; c) DLS size distribution of the same sample. Measurements were performed at 25°C.
  • Figure 3 Variation of the average size ( ⁇ ) and PDI (o) of a PEC0.&2 with temperature. Increments of 2°C were applied, with 10 minutes stabilization time at each temperature before the size distribution was measured. The crossed symbols correspond to the measurement at 25°C after the heating cycle.
  • FIG. 6 AFM images (height, tapping mode) of TMC/cc-GAL PEC0.S2 with TPP (TMC/TPP mass ratio: 3.6), on an HOPG substrate: a) in buffer HEPES 10 mM, pH 7.5; b) after pH was decreased to 3.9.
  • TMC 647N-labeled TMC
  • the present invention relates to a polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm.
  • polyelectolite complex is interchangeable used as "colloidal polyelectrolyte complex” or "PEC”.
  • the mass ratio protein/chitosan derivative is between 1 and 2.
  • Said complex presents very well controlled sizes with average particle 35 diameter below 200 nm and with a polidispersity (PDI) lower than 0.2.
  • PDI polidispersity
  • said polyelectrolyte complex is specially designed to carry proteins.
  • said protein is a lysosomal enzyme.
  • said lysosomal enzyme is a- galactosidase A (cc-GalA).
  • Chitosan is a natural polysaccharide is a natural linear polysaccharide composed of randomly distributed ⁇ -
  • a chitosan derivative is used since chitosan has many resources in nature, possesses a large number of reactive groups that makes its properties tuneable and is biocompatible and easily degradable in the human body, and preferably said derivative is N-trialkyl chitosan, more preferably wherein said
  • alkyl is methyl (TMC). Said alkylation is said to be “partial” since in the final product in addition to have trimethylated amines it is possible to find acetylated amines, dimethylated amines and even free amines. Therefore, where reference is made to "N- trialkyl chitosan” or “N-trimethyl chitosan” is to be understood that the alkylation is partial. In other words, “partially” in the present context means that chitosan is not
  • Chitosan and its derivatives are cationic polymers that can interact and form complexes with proteins that bear a negative charge at pH ⁇ pI.
  • the preferred protein used in the present invention, cc-GalA is a negatively charged enzyme (at neutral pH). Chitosan is only soluble in aqueous media and thus, positively charged,
  • the partial quatemization of the amine groups of chitosan diminishes the buffering capacity of the polyelectrolyte.
  • Polymers with high buffer capacity have been shown to escape from endosome via the mechanism called "proton sponge", 35 which ends up in lysis of the endosome, delivering the cargo to the cytoplasm. Therefore, the partial quaternization of chitosan is also intended to avoid this effect, as the protein is to be released preferably in the lysosomes.
  • TMC Due to the reactive groups of TMC, they can be optionally conjugated to specific ligands responsible for the interaction with integrins of certain cell membranes, in particular endothelial cells or specific for other tissues.
  • said conjugated ligand is a RGD derivative, i.e. a peptide containing the Arg-Gly-Asp (RGD) sequence, known to have a high binding affinity with integrins expressed in human vein endothelial cells.
  • RGD Arg-Gly-Asp
  • Chitosan derivative in particular TMC
  • TMC can also be functionalized with a fluorochrome in order to subsequently obtain fluorescently labelled TMC/protein PECs to carry on in vitro cellular up-take studies.
  • said chitosan derivative is ionically crosslinked with the third component, i.e. tripolyphosphate (also known as TPP).
  • TPP tripolyphosphate
  • TMC TPP
  • protein TPC
  • the present invention also relates to a process for manufacturing the polyelectrolyte complex according to any of the previous embodiments, comprising the steps of preparing solutions with PEC components (TMC, TPP and protein) in suitable buffer conditions and mixing the chitosan derivative solution with a solution containing the protein and tripolyphosphate polyanion.
  • said protein is a lysosomal enzyme.
  • said lysosomal enzyme is cc-galactosidase A (cc-GalA).
  • the obtained average particle diameter size is lower than 200 nm, particularly less than 160 nm (depending on the ratio of chitosan derivative (in particular, TMC/enzyme) with a polydispersity index (PDI) lower than 0.2. They have a positive Z potential. These PECs are very stable and do not aggregate over time or temperature up to 50°C.
  • the PECs were designed in order to be capable to survive, after internalized by the target cells, until the late endosome or the lysosome, where the acidic pH (pH in the late endosome is around 5.5, and in the lysosome, 4.5-5) would trigger the decomplexation and protein release.
  • the pH drop to the value correspondent to the late endosome/lysosome was shown to disassemble the TMC/protein PECs structure, and as a consequence, releasing the enzyme.
  • TMC/protein PECs obtained were stable in cell culture media without serum and internalized by cells from the human microvascular endothelial cell line (HMEC-1) after a short time of incubation at 37°C. After about 10-15 minutes of incubation some degree of co-localization with the lysosomes was already observed.
  • HMEC-1 human microvascular endothelial cell line
  • TMC/protein PECs arrive to the lysosomes, and, as the pH there is around 4.5, the enzyme is liberated in the desired intracellular compartment.
  • the present invention also relates to the use of the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention as a carrier and delivery system for proteins.
  • said system is delivered to lysosomes. More preferably, said lysosomes are in endothelial cells.
  • the present invention also relates to the use of the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for the manufacture of a medicament for treating a lysosomal disease.
  • said lysosomal disease is Fabry disease.
  • the present invention also relates to the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for use as a carrier and delivery system for proteins.
  • said system is delivered to lysosomes.
  • said lysosomes are in endothelial cells.
  • the present invention further relates to a polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for use in treating a lysosomal disease.
  • said lysosomal disease is Fabry disease.
  • the present invention also relates to a method of treatment of a subject with a lisosomal disease comprising admisnitering a polyelectrolyte complex and all its possible embodiments according to the present invention to a patient in need thereof.
  • a lisosomal disease comprising admisnitering a polyelectrolyte complex and all its possible embodiments according to the present invention to a patient in need thereof.
  • said lysosomal disease is Fabry disease.
  • the present invention further relates to a medicament comprising the polyelectrolyte complex in all its possible embodiments disclosed in the present invention.
  • TMC N-trimethyl chitosan
  • the polymer was precipitated with ethanol, isolated by centrifugation and thoroughly washed with ethanol and ether. In vacuum drying yielded a white, water- soluble product. Finally, it was dissolved in water and freeze-dried (freeze dryer ALPHA 1-4 LD, Christ).
  • the presence of ternary and quaternary amines in the TMC obtained was verified by 1H nuclear magnetic resonance (1H NMR).
  • the molecular weight of the TMC was estimated via gel permeation chromatography (GPC), using Ultrahydrogel 250 and 500 columns and a Waters 24/4 IR detector, in acetate buffer 0.5 M, pH 3. Dextran standards were employed for calibration.
  • TMC was characterized by pH-potentiometric titration to evaluate its buffer capacity. For this, 8 mg of the TMC were dissolved in 0.4 ml of hydrochloric acid (HC1) 0.3 M solution. Then, a solution of NaOH 0.06 M was used for titration while the pH of the solution was monitored with a calibrated pH-sensitive glass electrode.
  • HC1 hydrochloric acid
  • Recombinant cc-GAL obtained with the plasmid pOpinE-GLA expressed in the cell line HEK 293F was kindly supplied by Dr. J. L. Corchero and Prof. A. Villaverde, from the Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona (IBB, UAB).
  • the polyelectrolyte complexes (PECs) between a-GAL and TMC (+ penta- sodium triphosphate, TPP) were prepared by adding a solution of TMC (in buffer HEPES 10 mM, pH 7.4-7.5) at concentration of 35 or 50 ⁇ g ml "1 to a solution 100 ⁇ g ml "1 in a-GAL and 23 ⁇ g ml "1 in TPP (in buffer HEPES 10 mM, pH 7.4) in a volume ratio 70:30, followed by vortex mixing for 5 s and 60 min incubation at room temperature. Afterwards the PECs were mantained at 4°C.
  • the TMC/cc-GAL PECs were visualized by atomic force microscopy (AFM) (Dimension 3100, Nanoscope IV controller, Veeco, Digital Instruments, CA, USA).
  • AFM atomic force microscopy
  • the PECs were adsorbed onto highly ordered pyrolytic graphite (HOPG) and imaged under buffer HEPES 10 mM of pH 7.4.
  • HOPG highly ordered pyrolytic graphite
  • the experiments were performed in tapping mode under controlled liquid environment, with a V-shaped silicon nitride cantilever provided with a pyramidal tip (nominal spring constant 0.32 N m "1 , nominal tip radius 20 nm).
  • the enzymatic activity of cc-GAL was determined with a fluorometric assay.
  • 25 ⁇ of the sample cc-GAL or PEC -without any separation step from the uncomplexed TMC and/or enzyme-, diluted in buffer acetate 10 mM, pH 4.5 to 1 :500, 1 : 1000, 1 :2000 and 1 :4000
  • buffer acetate 10 mM, pH 4.5 to 1 :500, 1 : 1000, 1 :2000 and 1 :4000 were incubated with 100 ⁇ of 4- methylumbelliferyl- -D-galactopiranoside solution 2.46 mM in buffer acetate 10 mM pH 4.5, in a bath at 37°C with constant shaking during 1 hour.
  • the reaction was stopped by the addition of 1.25 ml of glycine buffer 100 mM of pH 10.4.
  • a calibration curve was obtained with 4-MU standard solutions in the glycine buffer ranging from 0-250 ng ml "1 .
  • the enzymatic activity was expressed as ⁇ ⁇ ⁇ 1(4- ⁇ ) nig(a-GAL) -1 h "1 .
  • TMC was conjugated to the florescent dye Atto 647N NHS ester.
  • Atto 647N NHS was first dissolved in a small amount of dimethyl sulfoxide (DMSO) and then diluted with buffer HEPES 10 mM pH 7.4. This solution was added to a TMC solution (in HEPES 10 mM pH 7.4) in an approximate molar ratio of 3: 1, and left to react for 1 h at room temperature under magnetic stirring.
  • Atto 647N-conjugated TMC (Atto647N-TMC) was separated from the free dye using Zeba desalt spin columns (Thermo Scientific).
  • a second conjugation step was performed with freshly prepared Atto 647N
  • the fluorescently-labeled TMC was purified with Zeba desalt spin columns as described above.
  • the Atto647N-TMC solution was freeze-dried (freeze dryer VirTis Freezemobile 25L).
  • the amount of conjugated dye was estimated to be 0.32 moles dye per mol TMC, via UV-visible spectrometry (NanoDrop Spectrophotometer ND-1000).
  • PECs were prepared mixing a solution of unlabeled TMC and Atto647N-TMC (in buffer HEPES 10 mM, pH 7.4-7.5) in a volume ratio 70:30 with the cc-GAL/TPP solution in the same buffer, as described in section 1.2.
  • HMEC-1 Human microvascular endothelial cell line, CDC/EU.HMEC-1 (HMEC-1), obtained from Centers for Disease Control and Prevention/National Center for Infectious Diseases (CDC-NIDR), was kindly provided by Dr. Ibane Abasolo (Hospital Univ. Vail d ' Hebron/CIBBIM, Barcelona). HMEC-1 cells were cultured in MCDB 131 (Invitrogen) with 50 units ml "1 penicillin, 50 ⁇ g ml "1 streptomycin, 10 mM L-Glutamine and supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% C0 2 at 37°C. All the media, sera and antibiotics were purchased from Invitrogen.
  • FBS fetal bovine serum
  • HMEC-1 cells were seeded at 2xl0 5 cells ml "1 36-48 h prior to experiment.
  • DPBS Dulbecco ' s phosphate buffered saline
  • MCDB 131 supplemented with 10 mM L-Glutamine without FBS.
  • Approximately 10 5 cells were incubated with 15 ⁇ g ml "1 Atto647-PECs for 2 hours at 16°C or 37°C.
  • Cells were subsequently washed twice with DPBS, resuspended in 300 ⁇ DPBS and subjected to FACS analysis. Data acquisition and analysis was performed using FACScan (Beckton-Dickinson) and BD FACSDiva software. 10000 viable cells were evaluated in each experiment and the results are the mean of 3 independent measurements.
  • HMEC-1 cells were seeded onto Fluorodish culture plates (World
  • the present inventors have developed a polyeletrolyte which has optimal properties for its self-assembly with the protein and further effective release in the target intracellular compartment (lysosome).
  • a-GAL is a homodimeric glycoprotein and it is markedly negatively charged.
  • the overall charge per monomer is expected to be -11 at neutral pH [2], and its pi is around 5.7. Therefore, in order to have a suitable polyelectrolyte able to form complexes with a-GAL, we prepared partially quaternized chitosan with the simplest alkyl group (methyl) (trimethyl chitosan, TMC), with ca. 50-60 % of quaternary amines (trymethylated amines) and 28 % degree of primary amines (free amines), as determined by l H NMR.
  • the molecular weight of the TMC was determined to be 202000 g mol "1 with a polydispersity of 2.6, using GPC.
  • the product was perfectly soluble in both water and 10 mM HEPES buffer at pH 7.4-7.5.
  • the present inventors demonstrated that the synthesized TMC is able to complex a-GAL and form stable polyelectrolyte complexes (PECs) with an average size lower than 200 nm and a polydispersity index (PDI) lower than 0.2.
  • PECs polyelectrolyte complexes
  • TMC/a-GAL PECs were prepared by adding TMC solution at two different concentrations to the solution of a-GAL in a volume ratio of 70:30.
  • DLS average particle diameter and ⁇ -potential of the suspension by DLS.
  • the formation of relatively stable complexes between TMC and a-GAL was observed over the pH range from 7.3 to 8.0.
  • the electrostatic interaction between the negatively charged a-GAL and the positively charged TMC led to complexes of average diameter around 70 to 90 nm and PDI between 0.2 and 0.4 (see tables 1 and 2).
  • the suspensions showed a positive ⁇ -potential in the range between 15 and 23 mV.
  • TPP polianion penta-sodium triphosphate
  • the physicochemical properties such as particle size, shape, surface charge and composition have been shown to play a key role in the blood circulation, cellular uptake and subsequent cellular routing of polymeric nanoparticles [8-10].
  • An empirical hydrodynamic diameter for efficient cellular uptake and systematic delivery of nanop articulate material has been established to be up to 200 nm [10, 1 1].
  • the herein shown results indicate that a small addition of the polyanion TPP led to PECs with average diameter ⁇ 200 nm and low polydispersity, and that varying the TMC/protein ratio and amount of TPP these parameters can be accurately controled.
  • the TMC/cc-GAL PECs are nanoparticles spherical in shape, they are homogeneous in size and no aggregation is observed, as metrology, in their natural liquid environment.
  • a topographical image of the PECs (TMC/cc-GAL mass ratio of 0.82 and TMC/TPP mass ratio of 3.6) adsorbed onto HOPG is shown in figure 2a-b. This result is in agreement with their DLS size distribution (figure 2c).
  • PECs with TMC/a-GAL ratios of 0.82 and 1.17, and with the corresponding amount of TPP as in table 2 were prepared at pH 7.5. Their average particle diameter size and PDI were measured upon preparation and after 4 days stored at 4°C. As it can be seen in table 3, the average size and PDI of the TMC/a-GAL PECs did not change significantly over time and no evidence of aggregation or flocculation of the nanoparticles could be observed. We had observed already that after one day that the ⁇ -potential of the PECs arrived to an almost constant value for TMC/a-GAL mass ratios of 0.82 and higher.
  • PEC0.&2 the PECs with TMC/a-GAL mass ratio of 0.82 and TMC/TPP mass ratio of 3.6 (named PEC0.&2 from here on) are chosen for further studies, as they are the ones with the minimum amount of TMC and TPP which present constant ⁇ -potential value, great stability and very narrow size distribution with average size lower than 200 nm.
  • PEC0.&2 have a 48 weight % of protein.
  • the present inventors have surprisingly found that the proposed entrapment process retains the enzyme functionality. Protein function is dependent on a precise and fragile three-dimensional structure that has to be maintained when nanocarriers are used for protein delivery purposes. For this reason, the effect of the protein entrapment process into the PECs on the cc-GAL functionality was evaluated. The enzymatic activity of the CC-GAL, both free and the in the TMC/cc-GAL PECs was compared, measured at pH 4.5, the optimum pH of activity of CC-GAL.
  • Figure 4 shows the activity values of cc-GAL, cc-GAL premixed with TPP, and cc-GAL in PECs at two different TMC/cc-GAL ratios (0.82 and 1.17, both with TPP).
  • cc-GAL retains most of its enzymatic activity, since only a decrease of 10 and 15 % in the activity is observed for PEC0.&2 and PEC/./ 7, respectively.
  • this result might indicate that at pH 4.5 most of the protein has been dissociated from the PECs and release to the medium.
  • the preferred target of the herein presented protein delivery system is the lysosomal compartment within the cells. Hence, it was developed such that the change from physiological pH (around 7.4) to the endosomal/lysosomal pH (between 5.5 and 4.5) acts as a trigger for the release of the protein from the PEC.
  • TMC has a low degree of pH- dependency of positive charge density when compared with chitosan.
  • the pH affects in a greater extent the protein charge density (as it approaches pi) as well as the TPP charges.
  • strong electrostatic interactions are the most predominant molecular forces that hold the PEC assembly, the pH drop weakens the assembly and, as a consequence, releases the protein.
  • HMEC-1 is an immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells [40].
  • Atto 647N shows excellent photostability and brightness, making it an excellent dye for tracking nanoparticles in live cells (wich are seen in red colour under the fluorescence microscope).
  • a mixture of unlabeled TMC and Atto647N-TMC solutions (volume ratio of 70:30) was used to prepare the fluorescently-labeled TMC/a-GAL PECs (including TPP) in the way previously described. Taking particle average diameter, PDI and ⁇ -potential as assessment parameters, stable and uniform fluorescent TMC/a-GAL PECs could be prepared.
  • Atto647N-PEC0.S2 presented an average diameter of 145.7 nm and PDI of 0.109 (figure 7). Moreover, Atto647N-labeled TMC/a-GAL PECs possessed good colloidal stability in both water/HEPES and serum-free cell culture medium. No significant increase in particle size or PDI was evident in medium.
  • Atto647N-TMC/a-GAL PECs The uptake of Atto647N-TMC/a-GAL PECs by HMEC-1 cells was evidenced by confocal fluorescence microscopy and flow cytometry.
  • TMC In absence of an oppositely charged molecule (e.g. a-GAL), TMC does not form stable nanoparticles in aqueous media, but tend to aggregate in diverse structures heterogeneous in size.
  • HMEC-1 cells were incubated with Atto647N-TMC or Atto647N-labeled TMC/a-GAL PECs for 2 h at 37°C at a dose of 15 ⁇ g/ml.
  • flow cytometry measurements indicate that about 30% of the cells showed enhanced fluorescence when incubated with Atto647N-TMC/cc-GAL PECs at 37°C.
  • Atto647N-TMC-incubated cells showed remaining fluorescence in flow cytometry (figure 8, left).
  • a decrease in the fraction of positive cells by FACS was observed when cells had been pulsed with TMC/a-GAL PECs at 16°C, a temperature precluding endocytosis (figure 8).
  • MFI mean fluorescence
  • HMEC- 1 The enhanced uptake of Atto647N-labeled TMC/a-GAL PECs by HMEC- 1 was additionally verified by confocal fluorescence microscopy.
  • HMEC-1 were incubated with Atto647N-labeled TMC/a-GAL PECs for 2h at 37°C, fluorescent red dots (due to Atto 647N) were clearly observed inside the cells.
  • Atto647N-TMC barely showed a minimum fluorescent intensity inside the cell [confocal fluorescent images are not shown as they are meaningless in black and white]. This indicates that, while the random structure of TMC might prevent its entry into the cells, the physicochemical properties of TMC/a-GAL PECs might facilitate their binding and subsequent uptake into the cells.
  • Lysotracker green under fluorescence microscope
  • PECs red
  • PECs ionically crosslinked colloidal polyelectrolyte complexes
  • TMC ionically crosslinked colloidal polyelectrolyte complexes
  • cc-GAL lysosomal protein cc-GAL
  • a positive Z potential prevents PECs from aggregating over time.
  • a relatively high protein load around 50 wt %) was achieved and, additionally, these PECs presented a spherical morphology and were stable in a temperature range up to 50°C at least for short periods.

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Abstract

The present invention relates to a polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm. The present invention also relates to a process for manufacturing said polyelectrolyte complex. The present invention further relates to the use of said polyelectrolyte complex as a carrier and delivery sustem for proteins, in particular to be delivered to lysosomes and more particularly in endothelial cells. The present invention additionally relates to use of said polyelectrolyte complex for the manufacture of a medicament for treating a lysosomal disease, in particular Fabry disease.

Description

POLYELECTROLYTE COMPLEX, PROCESS FOR ITS MANUFACTURE
AND USE THEREOF
Field of the invention
The present invention relates to a carrier and delivery system for proteins, in particular a polyelectrolyte complex (PEC). The present invention particularly relates to a polyelectrolyte complex designed to release the protein in lysosomes.
Background of the invention
The enzyme replacement therapy (ERT) is an established strategy for treating lysosomal storage diseases that consists on the intravenous supplementation of deficient cells with the enzyme which is poor or absent. Enormous progress in research toward development of ERT has been made in the last decade [1].
Fabry disease is a rare metabolic chromosome X-linked lysosomal storage disorder caused by the deficiency of a-galactosidase A (a-Gal A or a-GAL), the enzyme responsible for the degradation of globotriaocylceramide (Gb3 or GL-3) and other neutral glycosphingolipids. This deficiency leads to the accumulation of incompletely degraded macromolecules in lysosomes, particularly in cells of the vascular endothelium. Lysosomal storage leads to distension of the intracellular organelle and subsequent cellular dysfunction that translates into the presentation of renal, cardiac and cerebrovascular manifestations [2, 3]. ERT with exogenously administrated recombinant a-GAL has been shown to effectively address some of the clinical complications shown associated with Fabry disease [1, 3-5]. However, this approach is not completely effective since the enzyme is partly removed from the circulation by the liver and the spleen.
Instead, nanoparticle-based carriers have gained increasing interest for the targeted delivery of therapeutics to the intracellular site of action. Among available approaches, polyelectrolyte complexes (PECs) formed by self-assambly between the therapeutic molecule (protein or DNA) and natural or synthetic polyelectrolytes into multimolecular particles, have found many applications due to their exclusive properties including mild preparation conditions by simple aqueous solution mixing at room temperature, and biocompatibility [6]. PECs are association colloids, a class of colloidal systems in which molecules reversibly associate into multimolecular particles with a well-defined subcellular size and structure [7]. The physicochemical characteristics (size, shape, charge, hydrophobicity, etc.) of nanoparticulate PEC species play an important role in the blood circulation, transport in cells and subsequent cellular routing [8-10]. (An empirical hydrodynamic diameter for efficient cellular uptake and systematic delivery of nanoparticulate material has been established to be up to 200 nm [10, 1 1]). Furthermore, the chosen polyelectrolyte should have optimal properties for self-assembly with the protein and effective release in the target intracellular compartment.
Natural polysaccharides have received more and more attention in the field of drug delivery, as they have various resources in nature, they possess a large number of reactive groups to make their properties tuneable, and many of them are biocompatible and easily degradable in the human body [12, 13]. Chitosan nanoparticles have been used as carrier in protein and gene delivery for a number of different applications [6, 14-26]. Its outstanding characteristics such as non-toxicity, biocompatibility, biodegradability and low cost make it one of the most widely used biopolymers in biomedical applications [27, 28]. Chitosan is a natural aminopolysaccharide, the second-most abundant polysaccharide after cellulose. Chitosan and its derivatives are cationic polymers (cationic polyelectrolytes) that can interact and form colloidal complexes with negatively charged biomacromolecules (DNA, proteins, polysaccharides) with nanoencapsulations purposes. However, its poor aqueous solubility and loss of penetration-enhancing activity above pH 6 are major drawbacks for its use at physiological conditions [29]. Quaternization of the amine groups of the chitosan molecule is one of the most explored strategies to overcome this drawback [30, 31]. Quaternized chitosan with the simplest alkyl group, trimethyl chitosan (TMC) has been used to complex and condense siRNA or DNA to yield polyplexes for gene delivery purposes [32-35] and to complex insulin for oral and nasal delivery [6, 14, 30,
36-38].
There is still then the need in the art of a protein delivery system with potential application as an alternative ERT for lysosomal disorder.
The present inventors have surprisingly found that by thoroughly tailoring the PECs components and their proportions, desirable properties (size, Zeta potential, colloidal stability and pH-dependence of their integrity) are achieved allowing the formation of stable vehicles without altering the protein functionality, as well as their internalization by cells and trafficking to the target organelles (lysosomes) via a pH- triggered mechanism. The first object of the present invention is to provide a polyelectrolyte complex with the characteristics to be used as a delivery and carrier system. In particular, said system is a carrier and delivery system for proteins and more particularly to be delivered in lysosomes in endothelial cells. These systems are to protect the protein structure from degradation or denaturisation in the blood stream and to enhance the efficiency via the triggered release in the specific intracellular compartment (lysosomes), when compared with the regular enzyme replacement therapies that use the bare protein or the protein conjugated to a director oligosaccharide.
The second object of the present invention is to provide a process for manufacturing said polyelectrolyte complex according to the first object of the invention in a very simple way without using organic solvents or extreme conditions that may endanger the protein structure.
The third object of the present invention is to use said PECs for manufacturing a medicament useful for treating a lysosomal disease, in particular Fabry disease.
Summary of the invention
The present invention relates to a polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm.
The present invention also relates to a process for manufacturing said polyelectrolyte complex.
The present invention further relates to the use of said polyelectrolyte complex as a carrier and delivery sustem for proteins, in particular to be delivered to lysosomes and more particularly in endothelial cells.
The present invention additionally relates to use of said polyelectrolyte complex for the manufacture of a medicament for treating a lysosomal disease, in particular Fabry disease.
Brief description of figures
Figure 1. pH-potentiometric titration curves of an acidified solution of chitosan (o) or
TMC (·), with NaOH.
Figure 2. a) AFM image (height, tapping mode) of a TMC/cc-GAL PEC in a mass ratio of 0.82 with TPP (TMC/TPP mass ratio: 3.6), on an HOPG substrate in buffer HEPES 10 mM, pH 7.5; b) profile from the AFM topographic image; c) DLS size distribution of the same sample. Measurements were performed at 25°C. Figure 3. Variation of the average size (·) and PDI (o) of a PEC0.&2 with temperature. Increments of 2°C were applied, with 10 minutes stabilization time at each temperature before the size distribution was measured. The crossed symbols correspond to the measurement at 25°C after the heating cycle.
5 Figure 4. Enzymatic activity in μιηο1(4-Μυ) nig(a-GAL)-1 h"1, for -GAL (with HEPES buffer), a-GAL as prepared for the complex formation (with TPP, HEPES buffer) and TMC/cc-GAL PECs (with TPP, see table 3) with mass ratio of 0.82 and 1.17. Activity measurements were taken five hours after PECs preparation.
Figure 5. a) DLS size distribution of TMC/cc-GAL PEC0.S2 with TPP (TMC/TPP 1 0 mass ratio: 3.6), in buffer HEPES 10 mM, at pH 7.5 and at pH 5.1 ; b) PDI of the same sample as a function of pH.
Figure 6. AFM images (height, tapping mode) of TMC/cc-GAL PEC0.S2 with TPP (TMC/TPP mass ratio: 3.6), on an HOPG substrate: a) in buffer HEPES 10 mM, pH 7.5; b) after pH was decreased to 3.9.
15 Figure 7. DLS size distribution of a PEC between TMC or TMC/Atto647N-TMC (70:30), and a-GAL in a mass ratio of 0.82 with TPP (TMC/TPP mass ratio: 3.6), in buffer HEPES 10 mM pH 7.5. For PEC0.S2: D=120.4 nm, D/=0.180; for Atto647- PEC0.S2: D=145.7 nm, D/=0.109.
Figure 8. Specific uptake of Atto 647N-labeled TMC/a-GAL PECO.82 (PEC) or Atto
2 0 647N-labeled TMC (TMC) in live HMEC-1. Flow cytometry quantitation of the fraction of cells that had internalized PECs as the percent (%) of Atto 647N-positive cells among the given number of cells counted (left); and mean fluorescence intensity (MFI) of Atto647N in the cells normalized to the maximum fluorescence intensity (n=3, mean ± SD) (right).
25
Description of the invention
The present invention relates to a polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm.
3 0 In the context of the present invention the term "polyelectolite complex" is interchangeable used as "colloidal polyelectrolyte complex" or "PEC".
In a prefered embodiment, the mass ratio protein/chitosan derivative is between 1 and 2.
Said complex presents very well controlled sizes with average particle 35 diameter below 200 nm and with a polidispersity (PDI) lower than 0.2. Protein component
Structurally, said polyelectrolyte complex is specially designed to carry proteins. In a preferred embodiment said protein is a lysosomal enzyme. In a more 5 preferred embodiment, said lysosomal enzyme is a- galactosidase A (cc-GalA).
Chitosan derivative component
Also in said complex a derivative of chitosan is used. Chitosan is a natural polysaccharide is a natural linear polysaccharide composed of randomly distributed β-
1 0 (l-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). In the present invention a chitosan derivative is used since chitosan has many resources in nature, possesses a large number of reactive groups that makes its properties tuneable and is biocompatible and easily degradable in the human body, and preferably said derivative is N-trialkyl chitosan, more preferably wherein said
15 alkyl is methyl (TMC). Said alkylation is said to be "partial" since in the final product in addition to have trimethylated amines it is possible to find acetylated amines, dimethylated amines and even free amines. Therefore, where reference is made to "N- trialkyl chitosan" or "N-trimethyl chitosan" is to be understood that the alkylation is partial. In other words, "partially" in the present context means that chitosan is not
2 0 100% alkylated in the amine group.
Chitosan and its derivatives are cationic polymers that can interact and form complexes with proteins that bear a negative charge at pH<pI. The preferred protein used in the present invention, cc-GalA, is a negatively charged enzyme (at neutral pH). Chitosan is only soluble in aqueous media and thus, positively charged,
25 only at acidic pH, when the amine groups are protonated. This is one of the reasons why we introduced a chemical modification to the chitosan macromolecules, in order to have a positively charged soluble molecule at a wider pH range, especially around 7.4. This modification consists of a partial quatemization of the free amine groups via a two-step procedure described in the literature [8]. Starting from a commercially
3 0 available chitosan, the modification leads to different degree of methylation.
On the other hand, in addition to increase the solubility in a wider pH range, the partial quatemization of the amine groups of chitosan diminishes the buffering capacity of the polyelectrolyte. Polymers with high buffer capacity have been shown to escape from endosome via the mechanism called "proton sponge", 35 which ends up in lysis of the endosome, delivering the cargo to the cytoplasm. Therefore, the partial quaternization of chitosan is also intended to avoid this effect, as the protein is to be released preferably in the lysosomes.
Due to the reactive groups of TMC, they can be optionally conjugated to specific ligands responsible for the interaction with integrins of certain cell membranes, in particular endothelial cells or specific for other tissues. In a preferred embodiment said conjugated ligand is a RGD derivative, i.e. a peptide containing the Arg-Gly-Asp (RGD) sequence, known to have a high binding affinity with integrins expressed in human vein endothelial cells. These ligands allow to obtain PECs with enhanced specificity to the vascular endothelium.
Chitosan derivative, in particular TMC, can also be functionalized with a fluorochrome in order to subsequently obtain fluorescently labelled TMC/protein PECs to carry on in vitro cellular up-take studies.
Tripolyphosphate polyanion component
In the polyelectrolyte complex, said chitosan derivative is ionically crosslinked with the third component, i.e. tripolyphosphate (also known as TPP).
All these components (TMC, TPP and protein) interact via ionic and other non-covalent interactions, which allow a pH dependence on the PEC structure.
The present invention also relates to a process for manufacturing the polyelectrolyte complex according to any of the previous embodiments, comprising the steps of preparing solutions with PEC components (TMC, TPP and protein) in suitable buffer conditions and mixing the chitosan derivative solution with a solution containing the protein and tripolyphosphate polyanion. In a preferred embodiment said protein is a lysosomal enzyme. In a more preferred embodiment, said lysosomal enzyme is cc-galactosidase A (cc-GalA).
Said mixing is preferably carried out at room temperature and under stirring. The obtained average particle diameter size is lower than 200 nm, particularly less than 160 nm (depending on the ratio of chitosan derivative (in particular, TMC/enzyme) with a polydispersity index (PDI) lower than 0.2. They have a positive Z potential. These PECs are very stable and do not aggregate over time or temperature up to 50°C.
In this way it was possible to obtain a PEC with spherical morphology (as observed by atomic force microscopy) with a TMC/protein mass ratio of about 0.5-1.0, and with only a small amount of TPP (TMC/TPP mass ratio around 2.5 to 5.0). During the TMC/protein PECs formation the protein retains most of the enzymatic activity when tested after encapsulation, at the pH 4.5 (pH at which the enzyme is active in the lysosomes). A decrease of only 10 to 15 % in the activity was observed, which may be associated with some remaining degree of association with the polysaccharide.
The PECs were designed in order to be capable to survive, after internalized by the target cells, until the late endosome or the lysosome, where the acidic pH (pH in the late endosome is around 5.5, and in the lysosome, 4.5-5) would trigger the decomplexation and protein release. The pH drop to the value correspondent to the late endosome/lysosome was shown to disassemble the TMC/protein PECs structure, and as a consequence, releasing the enzyme.
The TMC/protein PECs obtained were stable in cell culture media without serum and internalized by cells from the human microvascular endothelial cell line (HMEC-1) after a short time of incubation at 37°C. After about 10-15 minutes of incubation some degree of co-localization with the lysosomes was already observed.
This proves that the TMC/protein PECs arrive to the lysosomes, and, as the pH there is around 4.5, the enzyme is liberated in the desired intracellular compartment.
Another surprising advantage of the present PECs is its stability over time and the studied temperature range (4 to 50°C) as it will be shown in more detail in the experimental section.
For further details about experimental data, see experimental section below.
The present invention also relates to the use of the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention as a carrier and delivery system for proteins. Preferably, said system is delivered to lysosomes. More preferably, said lysosomes are in endothelial cells.
The present invention also relates to the use of the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for the manufacture of a medicament for treating a lysosomal disease.
Preferably said lysosomal disease is Fabry disease.
The present invention also relates to the polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for use as a carrier and delivery system for proteins. Preferably, said system is delivered to lysosomes. More preferably, said lysosomes are in endothelial cells. The present invention further relates to a polyelectrolyte complex of the present invention and all its possible embodiments according to the present invention for use in treating a lysosomal disease. Preferably said lysosomal disease is Fabry disease.
The present invention also relates to a method of treatment of a subject with a lisosomal disease comprising admisnitering a polyelectrolyte complex and all its possible embodiments according to the present invention to a patient in need thereof. Preferably said lysosomal disease is Fabry disease.
The present invention further relates to a medicament comprising the polyelectrolyte complex in all its possible embodiments disclosed in the present invention.
Examples
The following Examples are provided to explain and illustrate the present invention and are not intended to be limiting of the invention.
Experimental Section
1. Materials and Methods
1.1 Synthesis and characterization of N-trimethyl chitosan
N-trimethyl chitosan (TMC) was prepared from chitosan middle-viscous (CS, Fluka, approximately 87 % degree of deacetylation, i.e. primary amines, according to lH NMR characterization), via a two-step procedure [39]. Briefly, a mixture of 2 g of sieved chitosan, 4.8 g of sodium iodide (Nal), 1 1 ml of a 15% aqueous sodium hydroxide (NaOH) solution and 1 1.5 ml of methyl iodide (CH3I) in 80 ml of l-methyl-2-pyrrolidinone was stirred at 60°C for 1 h, and a Liebig condenser was used. The product was precipitated using ethanol and thereafter isolated by centrifugation. The N-trimethyl chitosan iodide obtained after this first step was washed twice with ether on a glass filter to remove the ethanol. It was then dissolved in 80 ml of l-methyl-2-pyrrolidinone and heated to 60°C, thus removing most of the absorbed ether. Subsequently, 4.8 g of Nal, 1 1 ml of 15% NaOH solution and 7 ml of CH3I were added with rapid stirring and the mixture was kept at 60°C for 30 min. Additional 2 ml of CH3I and 0.6 g of NaOH pellets were added and the stirring was continued for 1 h. The product, precipitated with ethanol as described above, was dissolved in 40 ml of a 10% sodium chloride (NaCl) aqueous solution to exchange the iodide. The polymer was precipitated with ethanol, isolated by centrifugation and thoroughly washed with ethanol and ether. In vacuum drying yielded a white, water- soluble product. Finally, it was dissolved in water and freeze-dried (freeze dryer ALPHA 1-4 LD, Christ).
The presence of ternary and quaternary amines in the TMC obtained was verified by 1H nuclear magnetic resonance (1H NMR). The molecular weight of the TMC was estimated via gel permeation chromatography (GPC), using Ultrahydrogel 250 and 500 columns and a Waters 24/4 IR detector, in acetate buffer 0.5 M, pH 3. Dextran standards were employed for calibration.
TMC was characterized by pH-potentiometric titration to evaluate its buffer capacity. For this, 8 mg of the TMC were dissolved in 0.4 ml of hydrochloric acid (HC1) 0.3 M solution. Then, a solution of NaOH 0.06 M was used for titration while the pH of the solution was monitored with a calibrated pH-sensitive glass electrode.
1.2 Polyelectrolyte complexes formation
Recombinant cc-GAL obtained with the plasmid pOpinE-GLA expressed in the cell line HEK 293F was kindly supplied by Dr. J. L. Corchero and Prof. A. Villaverde, from the Institute for Biotechnology and Biomedicine, Autonomous University of Barcelona (IBB, UAB).
The polyelectrolyte complexes (PECs) between a-GAL and TMC (+ penta- sodium triphosphate, TPP) were prepared by adding a solution of TMC (in buffer HEPES 10 mM, pH 7.4-7.5) at concentration of 35 or 50 μg ml"1 to a solution 100 μg ml"1 in a-GAL and 23 μg ml"1 in TPP (in buffer HEPES 10 mM, pH 7.4) in a volume ratio 70:30, followed by vortex mixing for 5 s and 60 min incubation at room temperature. Afterwards the PECs were mantained at 4°C.
1.3 Physicochemical characterization
The average size (D), polidispersity index (PDI) and Zeta pontential (ξ- potential) values were determined by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments) equipped with a He-Ne laser (λ=633 nm) as the incident beam, using folded capillary cells.
The TMC/cc-GAL PECs were visualized by atomic force microscopy (AFM) (Dimension 3100, Nanoscope IV controller, Veeco, Digital Instruments, CA, USA). For this, the PECs were adsorbed onto highly ordered pyrolytic graphite (HOPG) and imaged under buffer HEPES 10 mM of pH 7.4. The experiments were performed in tapping mode under controlled liquid environment, with a V-shaped silicon nitride cantilever provided with a pyramidal tip (nominal spring constant 0.32 N m"1, nominal tip radius 20 nm).
1.4 Enzymatic activity
The enzymatic activity of cc-GAL was determined with a fluorometric assay. In short, 25 μΐ of the sample (cc-GAL or PEC -without any separation step from the uncomplexed TMC and/or enzyme-, diluted in buffer acetate 10 mM, pH 4.5 to 1 :500, 1 : 1000, 1 :2000 and 1 :4000) were incubated with 100 μΐ of 4- methylumbelliferyl- -D-galactopiranoside solution 2.46 mM in buffer acetate 10 mM pH 4.5, in a bath at 37°C with constant shaking during 1 hour. The reaction was stopped by the addition of 1.25 ml of glycine buffer 100 mM of pH 10.4. A spectra fluorophotometer (RF-1050 Shimadzu) was used to read the fluorescence of the samples (^=365 nm, em=450 nm), corresponding to the 4-methylumbelliferone (4- MU) produced during the reaction. A calibration curve was obtained with 4-MU standard solutions in the glycine buffer ranging from 0-250 ng ml"1. The enzymatic activity was expressed as μιηο1(4-Μυ) nig(a-GAL)-1 h"1.
1.5 In vitro cellular uptake studies
1.5.1 Preparation of fluorescently-labeled PECs
TMC was conjugated to the florescent dye Atto 647N NHS ester. Atto 647N NHS was first dissolved in a small amount of dimethyl sulfoxide (DMSO) and then diluted with buffer HEPES 10 mM pH 7.4. This solution was added to a TMC solution (in HEPES 10 mM pH 7.4) in an approximate molar ratio of 3: 1, and left to react for 1 h at room temperature under magnetic stirring. Atto 647N-conjugated TMC (Atto647N-TMC) was separated from the free dye using Zeba desalt spin columns (Thermo Scientific). In order to increase the amount of covalently attached Atto 647N into TMC, a second conjugation step was performed with freshly prepared Atto 647N
NHS solution for another hour and, afterwards, the fluorescently-labeled TMC was purified with Zeba desalt spin columns as described above. The Atto647N-TMC solution was freeze-dried (freeze dryer VirTis Freezemobile 25L). The amount of conjugated dye was estimated to be 0.32 moles dye per mol TMC, via UV-visible spectrometry (NanoDrop Spectrophotometer ND-1000). The fluorescently-labeled
PECs were prepared mixing a solution of unlabeled TMC and Atto647N-TMC (in buffer HEPES 10 mM, pH 7.4-7.5) in a volume ratio 70:30 with the cc-GAL/TPP solution in the same buffer, as described in section 1.2.
1.5.2 Cell culture Human microvascular endothelial cell line, CDC/EU.HMEC-1 (HMEC-1), obtained from Centers for Disease Control and Prevention/National Center for Infectious Diseases (CDC-NIDR), was kindly provided by Dr. Ibane Abasolo (Hospital Univ. Vail d'Hebron/CIBBIM, Barcelona). HMEC-1 cells were cultured in MCDB 131 (Invitrogen) with 50 units ml"1 penicillin, 50 μg ml"1 streptomycin, 10 mM L-Glutamine and supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere with 5% C02 at 37°C. All the media, sera and antibiotics were purchased from Invitrogen.
1.5.3 Flow cytometry
HMEC-1 cells were seeded at 2xl05 cells ml"1 36-48 h prior to experiment.
Cells were detached using trypsin, washed with Dulbecco's phosphate buffered saline (DPBS) and resuspended into MCDB 131 supplemented with 10 mM L-Glutamine without FBS. Approximately 105 cells were incubated with 15 μg ml"1 Atto647-PECs for 2 hours at 16°C or 37°C. Cells were subsequently washed twice with DPBS, resuspended in 300 μΐ DPBS and subjected to FACS analysis. Data acquisition and analysis was performed using FACScan (Beckton-Dickinson) and BD FACSDiva software. 10000 viable cells were evaluated in each experiment and the results are the mean of 3 independent measurements.
1.5.4 Confocal laser scanning microscopy (CLSM).
HMEC-1 cells were seeded onto Fluorodish culture plates (World
Precision Instruments, Sarasota, FL) at a density of 2xl04 cells plate"1 and allowed to grow for 36-48 hours. Fluorescently-labeled PECs were incubated with the cells for 0.5 to 3 h at 37°C and 5% C02, in serum-free MCDB 131 supplemented with 10 mM L-Glutamine. Subsequently, cells were washed three times with serum-free MCDB 131 and incubated at 37°C for 10 min with Lysotracker Green DND-26 (50 nM,
Molecular probes, Eugene, Oregon) to label the endosomal/lysosomal compartments. The nuclei in live cells were stained with Hoechst 33342 dye (Sigma). The cells were examined under an inverted Leica SP5 scanning confocal microscope (60x 1.42 NA oil immersion objective) fitted with a CCD camera. Data acquisition and analysis was performed with LAS-AF sotware and ImageJ.
2. Results
2.1 TMC synthesis and characterization The present inventors have developed a polyeletrolyte which has optimal properties for its self-assembly with the protein and further effective release in the target intracellular compartment (lysosome).
a-GAL is a homodimeric glycoprotein and it is markedly negatively charged. The overall charge per monomer is expected to be -11 at neutral pH [2], and its pi is around 5.7. Therefore, in order to have a suitable polyelectrolyte able to form complexes with a-GAL, we prepared partially quaternized chitosan with the simplest alkyl group (methyl) (trimethyl chitosan, TMC), with ca. 50-60 % of quaternary amines (trymethylated amines) and 28 % degree of primary amines (free amines), as determined by lH NMR. The molecular weight of the TMC was determined to be 202000 g mol"1 with a polydispersity of 2.6, using GPC. The product was perfectly soluble in both water and 10 mM HEPES buffer at pH 7.4-7.5.
The pH-potentiometric titration curves of chitosan (CS) and TMC acidified solutions, with a strong base (NaOH) (Figure 1) exhibit two inflection points. The first one is due to the neutralization of excess HC1, while the second one is ascribed to the neutralization of the protonated amine groups, and the difference in volume of NaOH between these two points is related with the buffer capacity of the polymer. As shown in the resulting curves in figure 1, TMC acid solution requires a higher amount of NaOH to neutralize the free acid, as there are less free amine groups to be protonated due to quartemization, and much less to neutralize the protonated amino groups
(AVcs=370 μΐ and AVTMC=75 μΐ). For TMC, a smaller percentage of the amines are protonable and the macromolecule's overall charge is positive, independently of the pH of the medium. Altogether, these data indicate that the buffer capacity of chitosan is strongly diminished after partial quaternization.
2.2 TMC/ -GAL PECs formation
The present inventors demonstrated that the synthesized TMC is able to complex a-GAL and form stable polyelectrolyte complexes (PECs) with an average size lower than 200 nm and a polydispersity index (PDI) lower than 0.2.
TMC/a-GAL PECs were prepared by adding TMC solution at two different concentrations to the solution of a-GAL in a volume ratio of 70:30. By means of monitoring the average particle diameter and ^-potential of the suspension by DLS, the formation of relatively stable complexes between TMC and a-GAL was observed over the pH range from 7.3 to 8.0. In this pH range, the electrostatic interaction between the negatively charged a-GAL and the positively charged TMC led to complexes of average diameter around 70 to 90 nm and PDI between 0.2 and 0.4 (see tables 1 and 2). The suspensions showed a positive ^-potential in the range between 15 and 23 mV. After storage at 4°C for one day, these PECs showed a slight increase in size and PDI, whereas the ^-potential values riched a relatively constant positive magnitude (24 to 29 mV) (table 1). However, the size distribution for these complexes was still considerably broad (PDI > 0.2).
Consequently, in order to enhance the stability and size homogeneity of the TMC/cc-GAL PECs, the polianion penta-sodium triphosphate (TPP) was introduced to induce some extent of ionic crosslinking, via the interaction with amines of different TMC molecules to give reversible and pH-dependent ionically crosslinked hydrogels.
The incorporation of TPP to the system led to an increase in the average size of the PECs (table 2). Nevertheless, and surprisingly, average particle diameter was always around 200 nm or lower and very narrow distributions were attained (PDI < 0.100), with no significant change in their ^-potential values. The PECs with TMC/cc-GAL mass ratios 0.82 and 1.17 both present a monomodal narrow size distribution, which suggests that all the protein and polysaccharide are involved in the PECs formation, i.e. loading of up to 1.2 ga-GAi/gTMC-
The physicochemical properties such as particle size, shape, surface charge and composition have been shown to play a key role in the blood circulation, cellular uptake and subsequent cellular routing of polymeric nanoparticles [8-10]. An empirical hydrodynamic diameter for efficient cellular uptake and systematic delivery of nanop articulate material has been established to be up to 200 nm [10, 1 1]. The herein shown results indicate that a small addition of the polyanion TPP led to PECs with average diameter < 200 nm and low polydispersity, and that varying the TMC/protein ratio and amount of TPP these parameters can be accurately controled.
Table 1. Average diameter (D), polydispersity index (PDI) and Zeta potential (ξ- potential) obtained by DLS at 25°C, for TMC/cc-GAL PECs in HEPES 10 mM pH 7.3, for TMC/cc-GAL mass ratios of 0.82 and 1.17, as prepared (to) and after 24 hs (tj).
TMC/a- to ~ 60 min ti ~ 24 hs
GAL mass
^-potential ξ- potential ratio D [nm] PDI D [nm] PDI
[mV] [mV]
0.82 86.94 0.191 18.4 ± 8.78 92.09 0.216 24.1 ± 5.96 1.17 72.55 0.245 22.9 ± 5.57 82.32 0.345 28.9 ± 5.22
Table 2. Average diameter (D), polydispersity index (PDI) and Zeta potential (ξ- potential) obtained by DLS at 25°C, for TMC/cc-GAL PECs in HEPES 10 mM pH 8.0, for TMC/cc-GAL mass ratios of 0.82 and 1.17, without and with TPP, as prepared.
TMC/a- without TPP with TPP
GAL
mass ^-potential TMC/TPP ^-potential
D [nm] PDI D [nm] PDI
ratio [mV] mass ratio [mV]
18.6 ±
0.82 76.76 0.365 21.3 ± 4.95 3.6 171.80 0.053
6.05
21.4 ±
1.17 63.04 0.390 17.0 ± 7.43 5.1 143.90 0.067
6.46
Furthermore, the TMC/cc-GAL PECs are nanoparticles spherical in shape, they are homogeneous in size and no aggregation is observed, as vizualized using AFM, in their natural liquid environment. As an example, a topographical image of the PECs (TMC/cc-GAL mass ratio of 0.82 and TMC/TPP mass ratio of 3.6) adsorbed onto HOPG is shown in figure 2a-b. This result is in agreement with their DLS size distribution (figure 2c).
2.3 Time and temperature stability of the TMC/ -GAL PECs
To evaluate their stability, PECs with TMC/a-GAL ratios of 0.82 and 1.17, and with the corresponding amount of TPP as in table 2, were prepared at pH 7.5. Their average particle diameter size and PDI were measured upon preparation and after 4 days stored at 4°C. As it can be seen in table 3, the average size and PDI of the TMC/a-GAL PECs did not change significantly over time and no evidence of aggregation or flocculation of the nanoparticles could be observed. We had observed already that after one day that the ^-potential of the PECs arrived to an almost constant value for TMC/a-GAL mass ratios of 0.82 and higher. One of the samples (TMC/a- GAL mass ratio 0.82) was left at 4°C and the average size and PDI were tested after 25 days. No significant variations from the original values were found (D=155.7 nm, D/=0.067). This means that the surface charge leading to the ^-potential values herein obtained, i.e. around 15 to 25 mV, is sufficient to provide electrostatic repulsion and prevent the particles from aggregating, at the tested conditions.
Following these results, the PECs with TMC/a-GAL mass ratio of 0.82 and TMC/TPP mass ratio of 3.6 (named PEC0.&2 from here on) are chosen for further studies, as they are the ones with the minimum amount of TMC and TPP which present constant ^-potential value, great stability and very narrow size distribution with average size lower than 200 nm. PEC0.&2 have a 48 weight % of protein.
Table 3. Average diameter (D), polydispersity index (PDI) and Zeta potential (ξ- potential) obtained by DLS at 25°C, for TMC/a-GAL PECs in HEPES 10 mM pH 7.5, for TMC/a-GAL mass ratios from 0.82 to 1.17, with TPP, as prepared (to) and after 4 days at 4°C (ti).
TMC/a-
TMC/TPP to ~ 60 min ti ~ 4 days GAL mass
mass ratio
ratio D [nm] PDI ξ-ροί. [mV] D [nm] PDI £-pot. [mV]
0.09
0.82 3.6 141.5 0.156 8.45 ± 6.56 141.2 20.3 ± 4.16
5
0.13
1.17 5.1 129.0 0.151 1 1.4 ± 8.30 129.2 20.0 ± 7.28
0
Furthermore, the stability of PEC0.S2 was demonstrated in the temperature range from 25 to 50°C. The average particle diameter size and PDI were recorded as the temperature was scanned. A continuous slight increase in the average particle diameter (and PDI) is observed while increasing temperature (figure 3), which is consistent with some extent of thermal expansion (reversible swelling). This result suggests that the colloidal PECs suspension is not destabilized in the temperature range of 25-50°C, and at a physiological temperature (37°C), only a 7% increment in the average particle diameter size is observed. Similar results were obtained when incubation time at 37°C was 1 h. Moreover, size and PDI values always remained within a satisfactory range (average diameter < 200 nm with PDI < 0.20). 2.4 Protein functionality after entrapment into PECs
The present inventors have surprisingly found that the proposed entrapment process retains the enzyme functionality. Protein function is dependent on a precise and fragile three-dimensional structure that has to be maintained when nanocarriers are used for protein delivery purposes. For this reason, the effect of the protein entrapment process into the PECs on the cc-GAL functionality was evaluated. The enzymatic activity of the CC-GAL, both free and the in the TMC/cc-GAL PECs was compared, measured at pH 4.5, the optimum pH of activity of CC-GAL. Figure 4 shows the activity values of cc-GAL, cc-GAL premixed with TPP, and cc-GAL in PECs at two different TMC/cc-GAL ratios (0.82 and 1.17, both with TPP). In both cases, cc-GAL retains most of its enzymatic activity, since only a decrease of 10 and 15 % in the activity is observed for PEC0.&2 and PEC/./ 7, respectively. Besides verifying that the structure of the protein is not altered by the encapsulation process, this result might indicate that at pH 4.5 most of the protein has been dissociated from the PECs and release to the medium.
2.5 pH-triggered protein release
The preferred target of the herein presented protein delivery system is the lysosomal compartment within the cells. Hence, it was developed such that the change from physiological pH (around 7.4) to the endosomal/lysosomal pH (between 5.5 and 4.5) acts as a trigger for the release of the protein from the PEC.
The effect of pH on the PECs structure and stability was assessed by monitoring the particle size distribution (average diameter and PDI) of a sample of PEC0.&2 while the pH was gradually dropped. At pH 7.5, this system has an average diameter of 155 nm with a PDI of 0.067 (see figure 5a). The particle average size and PDI remained unaltered until pH 6.8 was reached. Further pH decreases led to an increase in the average diameter and monomodal size distributions were no longer observed, with the consequent increase in PDI. At pH 5.1 the size distribution showeda mean value of 702.2 nm, with a PDI of 0.439 (figure 5a). The pH effect on the PECs structure is better noticed by plotting the PDI against pH. As shown in figure 5b, an inflexion in the PDI-pH curve occurs around pH 6.7. TMC has a low degree of pH- dependency of positive charge density when compared with chitosan. However, the pH affects in a greater extent the protein charge density (as it approaches pi) as well as the TPP charges. As strong electrostatic interactions are the most predominant molecular forces that hold the PEC assembly, the pH drop weakens the assembly and, as a consequence, releases the protein.
In order to vizualize this effect, an in situ AFM inspection of the PECs adsorbed onto HOPG was performed while pH was decreased from 7.5 to 3.9. The spherical nanoparticulate PECs are immediately disassembled upon a pH drop from
7.5 to 3.9 (figure 6).
Following these observations, it is expected that the hydrogel network forming the TMC/a-GAL PECs be already disrupted at pH 5.5, the pH of the endosomal compartment, and, as a result, the PECs components follow the path to the lysosomes, where the TMC would be enzymatically degraded and cc-GAL would exert its action.
2.6 Cellular uptake studies of TMC/a-GAL PECs
The major clinical manifestations of Fabry disease are belived to be caused by a progressive accumulation of Gb3 and other glycosphingolipids in vascular endothelial cells. Then, these cells represent the target of choice for therapeutical intervention. In order to assess the uptake of TMC/a-GAL PECs into endothelial cells we used HMEC-1 , which is an immortalized human microvascular endothelial cell line that retains the morphologic, phenotypic, and functional characteristics of normal human microvascular endothelial cells [40].
To track the uptake and distribution of the TMC/a-GAL PECs in these cells, we first engineered a-GAL-containing PECs from TMC that was labeled with Atto 647N. Atto 647N shows excellent photostability and brightness, making it an excellent dye for tracking nanoparticles in live cells (wich are seen in red colour under the fluorescence microscope). A mixture of unlabeled TMC and Atto647N-TMC solutions (volume ratio of 70:30) was used to prepare the fluorescently-labeled TMC/a-GAL PECs (including TPP) in the way previously described. Taking particle average diameter, PDI and ^-potential as assessment parameters, stable and uniform fluorescent TMC/a-GAL PECs could be prepared. The fluorescently-labeled PECs
(Atto647N-PEC0.S2) presented an average diameter of 145.7 nm and PDI of 0.109 (figure 7). Moreover, Atto647N-labeled TMC/a-GAL PECs possessed good colloidal stability in both water/HEPES and serum-free cell culture medium. No significant increase in particle size or PDI was evident in medium.
The uptake of Atto647N-TMC/a-GAL PECs by HMEC-1 cells was evidenced by confocal fluorescence microscopy and flow cytometry. We first compared the uptake efficiency of Atto647N-PEC0.S2 with that of their precursor, the Atto647N-TMC. In absence of an oppositely charged molecule (e.g. a-GAL), TMC does not form stable nanoparticles in aqueous media, but tend to aggregate in diverse structures heterogeneous in size. HMEC-1 cells were incubated with Atto647N-TMC or Atto647N-labeled TMC/a-GAL PECs for 2 h at 37°C at a dose of 15 μg/ml. As shown in figure 8 (left), flow cytometry measurements indicate that about 30% of the cells showed enhanced fluorescence when incubated with Atto647N-TMC/cc-GAL PECs at 37°C. Unlikely, only about 1% of the Atto647N-TMC-incubated cells showed remaining fluorescence in flow cytometry (figure 8, left). Furthermore, a decrease in the fraction of positive cells by FACS was observed when cells had been pulsed with TMC/a-GAL PECs at 16°C, a temperature precluding endocytosis (figure 8).
Even more importantly, the mean fluorescence (MFI) values measured for HMEC-1 incubated with Atto647N-labeled TMC/a-GAL PECs was 5-6 times higher than that of the cells incubated with Atto647N-TMC (figure 8, right).
The enhanced uptake of Atto647N-labeled TMC/a-GAL PECs by HMEC- 1 was additionally verified by confocal fluorescence microscopy. When HMEC-1 were incubated with Atto647N-labeled TMC/a-GAL PECs for 2h at 37°C, fluorescent red dots (due to Atto 647N) were clearly observed inside the cells. However, cells incubated with the Atto647N-TMC barely showed a minimum fluorescent intensity inside the cell [confocal fluorescent images are not shown as they are meaningless in black and white]. This indicates that, while the random structure of TMC might prevent its entry into the cells, the physicochemical properties of TMC/a-GAL PECs might facilitate their binding and subsequent uptake into the cells.
Our results show that a significant proportion of the cell-associated PECs were distributed within the cells. This internalization of PECs could not be attributed to a disruption of the cell plasma membrane since the dose of PECs did not alter cell viability. Then, it is very likely that the PECs were internalized by adsorptive endocytosis, an energy-dependent process that is preceded by non-specific interaction of the cargo with the cell membrane [41].
In the context of ERT for Fabry disease, lysosomal destination of TMC/a- GAL PECs is desired. Then, we tested whether internalized PECs trafficked to endosomal/lysosomal compartments in live HMEC-1. To do that, we used Lysotracker (green under fluorescence microscope), a fluorescent probe which labels and tracks acidic organelles. This dye accumulates in lysosomes, staining them with green fluorescence; therefore, PECs (red) accumulating in these organelles produced a yellow color. Some punctuate yellow color was noted after only 10-15 min of incubation with Atto647-PECs but an important fraction of fluorescent PECs colocalized with lysosomes within 1 h [confocal fluorescent images are not shown as they are meaningless in black and white].
As shown in previous experiments (figures 5 and 6), acid pH destabilizes the TMC/cc-GAL PEC favouring the dissociation between cc-GAL and TMC. Nevertheless, the tailored polyelectrolyte, with low buffer capacity, prevents an increase in the osmolality of the endolysosomal compartment and, in turn, its subsequent rupture. Then, even if cc-GAL is dissociated from TMC in the endosome, no endosomal escape is expected to occur and PECs components reach the lysosome, where the TMC would be enzymatically degraded and cc-GAL would exert its action.
3. Conclusions
As shown above, the present inventors have successfully prepared ionically crosslinked colloidal polyelectrolyte complexes (PECs) between TMC and the lysosomal protein cc-GAL by self assembly with a very simple procedure. Advantageously, these PECs showed very good narrow size distribution with average diameter < 200 nm and PDI < 0.2, desired values for a proper cellular internalization and further lysosomal destination. A positive Z potential prevents PECs from aggregating over time. Also advantageously, a relatively high protein load (around 50 wt %) was achieved and, additionally, these PECs presented a spherical morphology and were stable in a temperature range up to 50°C at least for short periods. We verified that the encapsulation process did not significantly affect the enzymatic activity of the CC-GAL, ensuring the integrity of the protein during and after the procedure. Following a successful fluorescent dye functionalization of the PECs, cellular uptake by the HMEC-1 cell line was demonstrated in vitro, and with an important degree of lysosomal co-localization after short periods of time. It has been proved that a pH drop from physiological to endosomal/lysosomal pH, leads to the dissociation of PECs and release of the protein. The herein presented pH-triggered carrier and delivery system for proteins has great potential as a delivery strategy for lysosomal enzyme replacement therapy with improved efficiency. In turn, the modification of said PECs with specific ligands for cell membrane receptors gives the possibility of increasing the specificity of therapy to a particular tissue and of targeting a particular internalization pathway.
References 1. Beck, M., Therapy for lysosomal storage disorders . IUBMB Life, 2010. 62(1): p. 33-40.
2. Garman, S.C. and D.N. Garboczi, The molecular defect leading to Fabry disease: Structure of human OC-
5 Galactosidase . Journal of Molecular Biology, 2004. 337(2): p. 319-335.
3. Rombach, S.M., et al . , Vasculopathy in patients with Fabry disease: Current controversies and research directions . Molecular Genetics and Metabolism, 2010.
10 99 (2) : p. 99-108.
4. Eng, C.M., et al . , Safety and efficacy of recombinant human OC-Galactosidase A replacement therapy in Fabry's disease. New England Journal of Medicine, 2001. 345 (1) : p. 9-16.
15 5. Schaefer, R.M., A. Tylki-Szymanska, and M.J. Hilz,
Enzyme replacement therapy for Fabry disease : A systematic review of available evidence . Drugs, 2009. 69(16) : p. 2179-2205.
6. Jintapattanakit , A., et al . , Peroral delivery of 20 insulin using chitosan derivatives: A comparative study of polyelectrolyte nanocomplexes and nanoparticles .
International Journal of Pharmaceutics, 2007. 342(1-2): p. 240-249.
7. Cohen Stuart, M.A., Supramolecular perspectives in 25 colloid science. Colloid & Polymer Science, 2008. 286(8-
9) : p. 855-864.
8. Moghimi, S.M., A.C. Hunter, and J.C. Murray, Nanomedicine : current status and future prospects . FASEB J. , 2005. 19 (3) : p. 311-330.
30 9. He, C, et al . , Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials , 2010. 31(13): p. 3657-3666.
10. Re man, J., et al . , Size-dependent internalization
35 of particles via the pathways of clathrin- and caveolae- mediated endocytosis. Biochem. J., 2004. 377(1): p. 159- 169.
11. Panyam, J. and V. Labhasetwar, Biodegradable nanoparticles for drug and gene delivery to cells and
5 tissue. Advanced Drug Delivery Reviews, 2003. 55(3): p.
329-347.
12. Liu, Z., et al . , Polysaccharides-based nanoparticles as drug delivery systems . Advanced Drug Delivery Reviews, 2008. 60(15): p. 1650-1662.
10 13. Boddohi, S. and M.J. Kipper, Engineering nanoassemblies of polysaccharides . Advanced Materials, 2010. 22 (28) : p. 2998-3016.
14. Amidi, M., et al . , Chitosan-based delivery systems for protein therapeutics and antigens. Advanced Drug
15 Delivery Reviews, 2010. 62(1): p. 59-82.
15. Prego, C, D. Torres, and M.J. Alonso, Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on the in vitro behaviour and in vivo effectiveness . Journal of
20 Nanoscience and Nanotechnology, 2006. 6(9-10): p. 2921- 2928.
16. Sarmento, B., et al . , Probing insulin's secondary structure after entrapment into alginate/chitosan nanoparticles . European Journal of Pharmaceutics and
25 Biopharmaceutics , 2007. 65(1): p. 10-17.
17. Tei eiro-Osorio, D., C. Remunan-Lopez , and M.J. Alonso, New generation of hybrid poly/oligosaccharide nanoparticles as carriers for the nasal delivery of macromolecules . Biomacromolecules , 2009. 10 (2): p. 243-
30 249.
18. Mao, S., et al . , Self-assembled polyelectrolyte nanocomplexes between chitosan derivatives and insulin. Journal of Pharmaceutical Sciences, 2006. 95(5): p. 1035- 1048. 19. Csaba, N., M. Koping-Hoggard, and M.J. Alonso, Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery. International Journal of Pharmaceutics, 2009.
5 382 (1-2) : p. 205-214.
20. Janes, K.A., P. Calvo, and M.J. Alonso, Polysaccharide colloidal particles as delivery systems for macromolecules . Advanced Drug Delivery Reviews, 2001. 47 (1) : p. 83-97.
10 21. Koping-Hoggard, M., et al . , Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo. Gene Therapy, 2001. 8(14): p. 1108-1121.
15 22. Liu, W.G. and K.D. Yao, Chitosan and its derivatives-A promising non-viral vector for gene transfection . Journal of Controlled Release, 2002. 83(1): p. 1-11.
23. Mao, S., W. Sun, and T. Kissel, Chitosan-based 20 formulations for delivery of DNA and siRNA. Advanced Drug
Delivery Reviews, 2010. 62(1): p. 12-27.
24. Thibault, M., et al . , Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes . Mol Ther, 2010.
25 25. Liu, W., et al . , An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes. Biomaterials , 2005. 26(15): p. 2705-2711.
26. Luppi, B., et al . , Chitosan-based hydrogels for nasal drug delivery: from inserts to nanoparticles . Expert
30 Opinion on Drug Delivery, 2010. 7(7): p. 811-828.
27. Jayakumar, R., et al . , Biomedical applications of chitin and chitosan based nanomaterials-A short review. Carbohydrate Polymers, 2010. 82(2): p. 227-232. 28. Rinaudo, M., Chitin and chitosan: Properties and applications. Progress in Polymer Science, 2006. 31(7): p. 603-632.
29. Bayat, A., et al . , Nanoparticles of quaternized 5 chitosan derivatives as a carrier for colon delivery of insulin: Ex vivo and in vivo studies . International Journal of Pharmaceutics, 2008. 356(1-2): p. 259-266.
30. Mourya, V.K. and N.N. Inamdar, Trimethyl chitosan and its applications in drug delivery. Journal of
10 Materials Science: Materials in Medicine, 2009. 20(5): p.
1057-1079.
31. Verheul, R.J., et al . , Synthesis, characterization and in vitro biological properties of O-methyl free Ν,Ν,Ν- trimethylated chitosan. Biomaterials , 2008. 29(27): p.
15 3642-3649.
32. Dehousse, V., et al . , Development of pH-responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery. Biomaterials, 2010. 31(7): p. 1839-1849.
20 33. Liu, W.G., et al . , N-Alkylated Chitosan as a
Potential Nonviral Vector for Gene Transfection . Bioconjugate Chemistry, 2003. 14(4): p. 782-789.
34. Thanou, M., et al . , Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines.
25 Biomaterials, 2002. 23(1): p. 153-159.
35. Germershaus, 0., et al . , Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft- trimethyl chitosan block copolymers : Establishment of structure-activity relationships in vitro. Journal of
30 Controlled Release, 2008. 125(2): p. 145-154.
36. Amidi, M., et al . , Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system. Journal of Controlled Release, 2006. 111(1-2): p. 107-116. 37. Yin, L., et al . , Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery. Biomaterials ,
2009. 30 (29) : p. 5691-5700.
38. Sonaje, K., et al . , Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-Responsive nanoparticles vs. subcutaneous injection . Biomaterials,
2010. 31 (26) : p. 6849-6858.
39. Sieval, A.B., et al . , Preparation and NMR characterization of highly substituted N-trimethyl chitosan chloride. Carbohydrate Polymers, 1998. 36(2-3): p. 157-165.
40. Ades, E.W., et al . , HMEC-1 : Establishment of an Immortalized Human Microvascular Endothelial Cell Line. J
Investig Dermatol, 1992. 99(6): p. 683-690.
41. Raub, T.J. and K.L. Audus, Adsorptive Endocytosis and Membrane Recycling by Cultured Primary Bovine Brain Microvessel Endothelial-Cell Monolayers . Journal of Cell Science, 1990. 97: p. 127-138.

Claims

I . - Polyelectrolyte complex comprising a protein, a chitosan derivative and tripolyphosphate polyanion with an average particle diameter size lower than 200 nm.
2.- Polyelectrolyte complex according to claim 1 , wherein the mass ratio protein/chitosan derivative is between 1 and 2.
3. - Polyelectrolyte complex according to any of claims 1 or 2 wherein said protein is a lysosomal enzyme.
4. - Polyelectrolyte complex according to any of the preceeeding claims wherein said lysosomal enzyme is cc-galactosidase A.
5. - Polyelectrolyte complex according to any of the preceeding claims wherein said chitosan derivative is N-trialkyl chitosan.
6. - Polyelectrolyte complex according to claim 5, wherein said N-trialkyl chitosan is N-trimethyl chitosan
7.- Polyelectrolyte complex according to any of the preceeding claims further comprising a conjugated ligand.
8. - Polyelectrolyte complex according to claim 7, wherein said conjugated ligand is a RGD derivative.
9. - Process for manufacturing the polyelectrolyte complex according to any of claims 1 to 8, comprising the step of mixing the chitosan derivative solution with a solution containing the protein and tripolyphosphate polyanion.
10. - Process according to the claim 9, wherein said protein is a lysosomal enzyme.
I I . - Process according to the claim 10, wherein said lysosomal enzyme is cc-galactosidase A.
12. - Process according to any of claims 9 to 1 1 , wherein said mixing is carried out at room temperature and under stirring.
13. - Use of the polyelectrolyte complex according to any of claims 1 to 8, as a carrier and delivery system for proteins.
14.- Use according to claim 13 wherein said system is delivered to lysosomes.
15. - Use according to claim 14 wherein said lysosomes are in endothelial cells.
16. - Use of the polyelectrolyte complex according to any of claims 1 to 8 for the manufacture of a medicament for treating a lysosomal disease.
17. - Use according to claim 16 wherein said lysosomal disease is Fabry disease.
18. - Polyelectrolyte complex according to any of claims 1 to 8 for use in treating a lysosomal disease.
19.- Polyelectrolyte complex according to claim 18 wherein said lysosomal disease is Fabry disease.
20.- Medicament comprising the polyelectrolyte complex according to any of claims 1-8 or 18-19.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021150997A3 (en) * 2020-01-22 2021-09-10 Engene, Inc. Localized expression of therapeutic nucleic acids in lung epithelial cells
US20230190667A1 (en) * 2015-08-13 2023-06-22 The Johns Hopkins University Methods of preparing polyelectrolyte complex nanoparticles

Non-Patent Citations (41)

* Cited by examiner, † Cited by third party
Title
ADES, E.W. ET AL.: "HMEC-l: Establishment of an Immortalized Human Microvascular Endothelial Cell Line", J INVESTIG DERMATOL, vol. 99, no. 6, 1992, pages 683 - 690, XP000885610, DOI: doi:10.1111/1523-1747.ep12613748
AMIDI, M. ET AL.: "Chitosan-based delivery systems for protein therapeutics and antigens", ADVANCED DRUG DELIVERY REVIEWS, vol. 62, no. 1, 2010, pages 59 - 82, XP026877915
AMIDI, M. ET AL.: "Preparation and characterization of protein-loaded N-trimethyl chitosan nanoparticles as nasal delivery system", JOURNAL OF CONTROLLED RELEASE, vol. 111, no. 1-2, 2006, pages 107 - 116, XP024957436, DOI: doi:10.1016/j.jconrel.2005.11.014
BAYAT, A. ET AL.: "Nanoparticles of quaternized chitosan derivatives as a carrier for colon delivery of insulin: Ex vivo and in vivo studies", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 356, no. 1-2, 2008, pages 259 - 266, XP022625196, DOI: doi:10.1016/j.ijpharm.2007.12.037
BECK, M.: "Therapy for lysosomal storage disorders", IUBMB LIFE, vol. 62, no. 1, 2010, pages 33 - 40
BODDOHI, S.; M.J. KIPPER: "Engineering nanoassemblies of polysaccharides", ADVANCED MATERIALS, vol. 22, no. 28, 2010, pages 2998 - 3016, XP055130855, DOI: doi:10.1002/adma.200903790
COHEN STUART, M.A.: "Supramolecular perspectives in colloid science", COLLOID & POLYMER SCIENCE, vol. 286, no. 8-9, 2008, pages 855 - 864, XP019628621
CSABA, N.; M. KOPING-HOGGARD; M.J. ALONSO: "Ionically crosslinked chitosan/tripolyphosphate nanoparticles for oligonucleotide and plasmid DNA delivery", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 382, no. 1-2, 2009, pages 205 - 214, XP026708561, DOI: doi:10.1016/j.ijpharm.2009.07.028
DEHOUSSE, V. ET AL.: "Development of pH-responsive nanocarriers using trimethylchitosans and methacrylic acid copolymer for siRNA delivery", BIOMATERIALS, vol. 31, no. 7, 2010, pages 1839 - 1849, XP026827545
ENG, C.M. ET AL.: "Safety and efficacy of recombinant human a-Galactosidase A replacement therapy in Fabry's disease", NEW ENGLAND JOURNAL OF MEDICINE, vol. 345, no. 1, 2001, pages 9 - 16
GARMAN, S.C.; D.N. GARBOCZI: "The molecular defect leading to Fabry disease: Structure of human a-Galactosidase", JOURNAL OF MOLECULAR BIOLOGY, vol. 337, no. 2, 2004, pages 319 - 335, XP004493187, DOI: doi:10.1016/j.jmb.2004.01.035
GERMERSHAUS, 0. ET AL.: "Gene delivery using chitosan, trimethyl chitosan or polyethylenglycol-graft- trimethyl chitosan block copolymers: Establishment of structure-activity relationships in vitro", JOURNAL OF CONTROLLED RELEASE, vol. 125, no. 2, 2008, pages 145 - 154, XP022402143
HE, C. ET AL.: "Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles", BIOMATERIALS, vol. 31, no. 13, 2010, pages 3657 - 3666, XP026946003, DOI: doi:10.1016/j.biomaterials.2010.01.065
JANES, K.A.; P. CALVO; M.J. ALONSO: "Polysaccharide colloidal particles as delivery systems for macromolecules", ADVANCED DRUG DELIVERY REVIEWS, vol. 47, no. 1, 2001, pages 83 - 97, XP055069619, DOI: doi:10.1016/S0169-409X(00)00123-X
JAYAKUMAR, R. ET AL.: "Biomedical applications of chitin and chitosan based nanomaterials-A short review", CARBOHYDRATE POLYMERS, vol. 82, no. 2, 2010, pages 227 - 232, XP027137630
JINTAPATTANAKIT, A. ET AL.: "Peroral delivery of insulin using chitosan derivatives: A comparative study of polyelectrolyte nanocomplexes and nanoparticles", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 342, no. 1-2, 2007, pages 240 - 249, XP022206329, DOI: doi:10.1016/j.ijpharm.2007.05.015
KOPING-HOGGARD, M. ET AL.: "Chitosan as a nonviral gene delivery system. Structure-property relationships and characteristics compared with polyethylenimine in vitro and after lung administration in vivo.", GENE THERAPY, vol. 8, no. 14, 2001, pages 1108 - 1121, XP002967348, DOI: doi:10.1038/sj.gt.3301492
LIU, W. ET AL.: "An investigation on the physicochemical properties of chitosan/DNA polyelectrolyte complexes.", BIOMATERIALS, vol. 26, no. 15, 2005, pages 2705 - 2711, XP025280694, DOI: doi:10.1016/j.biomaterials.2004.07.038
LIU, W.G. ET AL.: "N-Alkylated Chitosan as a Potential Nonviral Vector for Gene Transfection", BIOCONJUGATE CHEMISTRY, vol. 14, no. 4, 2003, pages 782 - 789, XP055274733, DOI: doi:10.1021/bc020051g
LIU, W.G.; K.D. YAO: "Chitosan and its derivatives-A promising non-viral vector for gene transfection", JOURNAL OF CONTROLLED RELEASE, vol. 83, no. 1, 2002, pages 1 - 11, XP004380054, DOI: doi:10.1016/S0168-3659(02)00144-X
LIU, Z. ET AL.: "Polysaccharides-based nanoparticles as drug delivery systems", ADVANCED DRUG DELIVERY REVIEWS, vol. 60, no. 15, 2008, pages 1650 - 1662, XP025656959, DOI: doi:10.1016/j.addr.2008.09.001
LUPPI, B. ET AL.: "Chitosan-based hydrogels for nasal drug delivery: from inserts to nanoparticles", EXPERT OPINION ON DRUG DELIVERY, vol. 7, no. 7, 2010, pages 811 - 828
MAO, S. ET AL.: "Self-assembled polyelectrolyte nanocomplexes between chitosan derivatives and insulin", JOURNAL OF PHARMACEUTICAL SCIENCES, vol. 95, no. 5, 2006, pages 1035 - 1048, XP002501783, DOI: doi:10.1002/JPS.20520
MAO, S.; W. SUN; T. KISSEL: "Chitosan-based formulations for delivery of DNA and siRNA", ADVANCED DRUG DELIVERY REVIEWS, vol. 62, no. 1, 2010, pages 12 - 27, XP026877912
MOGHIMI, S.M.; A.C. HUNTER; J.C. MURRAY: "Nanomedicine: current status and future prospects", FASEB J., vol. 19, no. 3, 2005, pages 311 - 330, XP009059925, DOI: doi:10.1096/fj.04-2747rev
MOURYA, V.K.; N.N. INAMDAR: "Trimethyl chitosan and its applications in drug delivery", JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE, vol. 20, no. 5, 2009, pages 1057 - 1079, XP019680201
PANYAM, J.; V. LABHASETWAR: "Biodegradable nanoparticles for drug and gene delivery to cells and tissue", ADVANCED DRUG DELIVERY REVIEWS, vol. 55, no. 3, 2003, pages 329 - 347, XP008096954, DOI: doi:10.1016/S0169-409X(02)00228-4
PREGO, C.; D. TORRES; M.J. ALONSO: "Chitosan nanocapsules as carriers for oral peptide delivery: Effect of chitosan molecular weight and type of salt on the in vitro behaviour and in vivo effectiveness", JOURNAL OF NANOSCIENCE AND NANOTECHNOLOGY, vol. 6, no. 9-10, 2006, pages 2921 - 2928
RAUB, T.J.; K.L. AUDUS: "Adsorptive Endocytosis and Membrane Recycling by Cultured Primary Bovine Brain Microvessel Endothelial-Cell Monolayers", JOURNAL OF CELL SCIENCE, vol. 97, 1990, pages 127 - 138
REJMAN, J. ET AL.: "Size-dependent internalization of particles via the pathways of clathrin- and caveolae- mediated endocytosis", BIOCHEM. J., vol. 377, no. 1, 2004, pages 159 - 169, XP055228473, DOI: doi:10.1042/bj20031253
RINAUDO, M.: "Chitin and chitosan: Properties and applications", PROGRESS IN POLYMER SCIENCE, vol. 31, no. 7, 2006, pages 603 - 632, XP025078628, DOI: doi:10.1016/j.progpolymsci.2006.06.001
ROMBACH, S.M. ET AL.: "Vasculopathy in patients with Fabry disease: Current controversies and research directions", MOLECULAR GENETICS AND METABOLISM, vol. 99, no. 2, 2010, pages 99 - 108, XP026835383
SARMENTO, B. ET AL.: "Probing insulin's secondary structure after entrapment into alginate/chitosan nanoparticles.", EUROPEAN JOURNAL OF PHARMACEUTICS AND BIOPHARMACEUTICS, vol. 65, no. 1, 2007, pages 10 - 17, XP005788720, DOI: doi:10.1016/j.ejpb.2006.09.005
SCHAEFER, R.M.; A. TYLKI-SZYMANSKA; M.J. HILZ: "Enzyme replacement therapy for Fabry disease: A systematic review of available evidence", DRUGS, vol. 69, no. 16, 2009, pages 2179 - 2205
SIEVAL, A.B. ET AL.: "Preparation and NMR characterization of highly substituted N-trimethyl chitosan chloride", CARBOHYDRATE POLYMERS, vol. 36, no. 2-3, 1998, pages 157 - 165, XP004134492, DOI: doi:10.1016/S0144-8617(98)00009-5
SONAJE, K. ET AL.: "Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: Oral delivery using pH-Responsive nanoparticles vs. subcutaneous injection.", BIOMATERIALS, vol. 31, no. 26, 2010, pages 6849 - 6858, XP027132793
TEIJEIRO-OSORIO, D.; C. REMUNAN-LOPEZ; M.J. ALONSO: "New generation of hybrid poly/oligosaccharide nanoparticles as carriers for the nasal delivery of macromolecules", BIOMACROMOLECULES, vol. 10, no. 2, 2009, pages 243 - 249, XP009138457, DOI: doi:10.1021/bm800975j
THANOU, M. ET AL.: "Quaternized chitosan oligomers as novel gene delivery vectors in epithelial cell lines", BIOMATERIALS, vol. 23, no. 1, 2002, pages 153 - 159, XP004322630, DOI: doi:10.1016/S0142-9612(01)00090-4
THIBAULT, M. ET AL.: "Intracellular trafficking and decondensation kinetics of chitosan-pDNA polyplexes", MOL THER, 2010
VERHEUL, R.J. ET AL.: "Synthesis, characterization and in vitro biological properties of O-methyl free N,N,N-trimethylated chitosan.", BIOMATERIALS, vol. 29, no. 27, 2008, pages 3642 - 3649, XP022849892, DOI: doi:10.1016/j.biomaterials.2008.05.026
YIN, L. ET AL.: "Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery", BIOMATERIALS, vol. 30, no. 29, 2009, pages 5691 - 5700, XP026469995, DOI: doi:10.1016/j.biomaterials.2009.06.055

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