Method of manufacturing peptide nanoparticles
Provided herein is a method of producing peptide nanoparticles comprising vortex-mixing (a) a hydrophobic or amphiphilic peptide or peptide conjugate, (b) one or more lipids that are free of a water-soluble polymer, (c) one or more lipids covalently attached to a water-soluble polymer, and (d) a hydrophilic solvent, to provide peptide nanoparticles.
1 . A method of producing high-loading peptide nanoparticles comprising vortex-mixing (a) a hydrophobic or amphiphilic peptide or peptide conjugate, (b) one or more lipids that are free of a water-soluble polymer, (c) one or more lipids covalently attached to a water-soluble polymer, and (d) a hydrophilic solvent, to provide high-loading peptide nanoparticles.
2 . The method of claim 1 , wherein the peptide nanoparticles comprise greater than about 30 mole % of the hydrophobic or amphiphilic peptide or peptide conjugate.
3 . The method of claim 1 , wherein the high-loading peptide nanoparticles comprise about 2 to about 20 mole % of the one or more lipids free of a water-soluble polymer.
4 . The method of claim 1 , wherein the high-loading peptide nanoparticles nanoparticles comprise about 10 to about 60 mole % of the one or more lipids covalently attached to a water-soluble polymer.
5 . The method claim 1 , wherein the hydrophobic or amphiphilic peptide or peptide conjugate is a peptide conjugated to a hydrophobic molecule or a lipidated peptide.
6 . The method of claim 1 , wherein the one or more lipids covalently attached to a water-soluble polymer comprise a lipid conjugated to a polyoxyethylene polymer.
7 . The method of claim 1 , wherein the vortex mixing comprises combining in a vortex mixer (a) an organic phase comprising (i) the hydrophobic or amphiphilic peptide or peptide conjugate, (ii) one or more lipids that are free of a water-soluble polymer, (iii) the one or more lipids covalently attached to a water-soluble polymer, and (iv) an organic solvent; and (b) an aqueous phase comprising a hydrophilic solvent, optionally water.
8 . The method of claim 1 , wherein the vortex mixing comprises combining (a) a first stream of an organic phase comprising (i) the hydrophobic or amphiphilic peptide or peptide conjugate, (ii) one or more lipids that are free of a water-soluble polymer, (iii) the one or more lipids covalently attached to a water-soluble polymer, and (iv) an organic solvent; and (b) a second stream of an aqueous phase comprising a hydrophilic solvent, optionally, water; wherein the first and second streams are combined under conditions sufficient to create a vortex.
9 . The method of claim 7 , wherein the organic solvent comprises an alcohol.
10 . The method of claim 7 , wherein the organic phase and aqueous phase are combined at a ratio of 1:5 to 1:50 in the vortex mixer.
11 . The method of claim 1 , wherein the vortex mixing is performed in a multiple-inlet vortex mixer.
12 . The method of claim 1 , wherein the method further comprises drying the peptide nanoparticles to provide a powder composition comprising the peptide nanoparticles.
13 . The method of claim 12 , wherein drying the peptide nanoparticles comprises lyophilization or spray drying.
14 . The method of claim 12 , wherein the method comprises adding a drying protectant prior to drying the nanoparticles.
15 . The method of claim 14 , wherein the protectant comprises trehalose, leucine, or combination thereof.
16 . The method of claim 1 , wherein the amphiphilic or hydrophobic peptide or peptide conjugate comprises FEEERI (SEQ ID NO: 1), FEKEKI (SEQ ID NO: 2), FEKERI (SEQ ID NO: 3), RGT (SEQ ID NO: 4), EEERA (SEQ ID NO: 5), FEEERA (SEQ ID NO: 6), FEEERM (SEQ ID NO: 7), FEEERL (SEQ ID NO: 8), FEKEKM (SEQ ID NO: 9), FEKEKL (SEQ ID NO: 10), FEKERM (SEQ ID NO: 11), FEKERL (SEQ ID NO: 12), CFEEERAC (SEQ ID NO: 13), FEEERAR (SEQ ID NO: 14), FEEERARA (SEQ ID NO: 15), SIRYSGHpSL (SEQ ID NO: 16), KFEEERARAKWDT (SEQ ID NO: 17), RCLLPA (SEQ ID NO: 18), or LLARRPTKGIHEY (SEQ ID NO: 19) optionally conjugated to a hydrophobic moiety.
17 . The method of claim 1 , wherein the amphiphilic or hydrophobic peptide or peptide conjugate comprises myr-FEEERL (SEQ ID NO: 8), myr-FEEERA (SEQ ID NO: 6), myr-FEKEKL (SEQ ID NO: 10), myr-FEEERM (SEQ ID NO: 7), myr-FEKERM (SEQ ID NO: 11), myr-FEKERL (SEQ ID NO: 12), myr-FEKERI (SEQ ID NO: 3), myr-CFEEERAC (SEQ ID NO: 13), and myr-SIRYSGH (p) SL.
18 . The method of claim 1 , wherein the vortex mixing comprises
(a) providing an organic phase comprising about 30-80 mole % of the hydrophobic or amphiphilic peptide, about 2-20 mole % of one or more lipids free of a water soluble polymer, and about 10-60 mole % of one or more lipid attached to a water soluble polymer dissolved in an organic solvent,
and (b) vortex-mixing the organic phase with an aqueous phase at about 5 to 50 times the volume of the organic phase.
19 . A method of preparing a peptide nanoparticle, the method comprising vortex mixing (a) an organic phase comprising an organic solvent and an amphiphilic peptide with (b) an aqueous phase, wherein the organic phase and aqueous phase do not comprise any free lipids;
or
comprising vortex-mixing (a) an organic phase comprising a hydrophobic or amphiphilic peptide or peptide conjugate and one of (i) one or more lipids that are free of a water-soluble polymer, or (ii) one or more lipids covalently attached to a water-soluble polymer, and (b) a hydrophilic solvent, to provide lipid-stabilized peptide nanoparticles,
wherein the amphiphilic peptide or peptide conjugate comprises myr-FEEERL (SEQ ID NO: 8), myr-FEEERA (SEQ ID NO: 6), myr-FEKEKL (SEQ ID NO: 10), myr-FEEERM (SEQ ID NO: 7), myr-FEKERM (SEQ ID NO: 11), myr-FEKERL (SEQ ID NO: 12), myr-FEKERI (SEQ ID NO: 3), myr-CFEEERAC (SEQ ID NO: 13), and myr-SIRYSGH(p)SL.