- Salt effects on polymers in aqueous solution
The lyotropic effects of salts on nonionic polymers (dextran1 and polyacrylamide2) in aqueous solutions and gels have been studied and interesting insights1, 2 regarding the impact on the osmotic pressure and on gel swelling were obtained.
The effects of a chaotropic salt, KSCN, on the lower critical solution temperature (LCST) of aqueous poly(n-isopropylacrylamide) (PNIPA) solutions.
To advance the understanding of the mechanism of salting-in salts effects, we have provided previously unreported calorimetric evidence for the binding of a chaotropic salt (KSCN) to poly-n-isopropylacrylamide (PNIPA), a model for proteins, using sensitive isothermal microcalorimetry, and provided a new explanation for the entropic binding mechanism3.
- Sugar effect on water structure and on polymer (particularly protein) stability
Studying the mechanisms of protection of saccharides against protein denaturation, and using PAAm4 and PNIPA as a models for certain aspects of protein solution behavior, we have shown a correlation between the hydration number of different sugar isomers and their effect on the phase transition temperature of PNIPA5, 6 and on the deswelling of PNIPA gel7. Using modeling, atomic molecular simulations, and advanced instrumental techniques, we have proposed and provided substantial support to a novel templating mechanism of saccharides on cooperative hydrogen bonding of their vicinal water. The better a sugar fits into an ideal water structure, as embodied in hexagonal ice, the better a template it will be, and consequently the higher its hydration number will be, and the stronger its protective effect against thermal denaturation of globular proteins8, 9.
- Casein micelles as nano-vehicles
We have introduced the potential of casein micelles to serve as nano-vehicles for added hydrophobic (and other) nutraceuticals (e.g. vitamin D) that can be used for enrichment of staple foods (mainly low or non-fat)10. Moreover, we found that the micelles can protect the encapsulated bioactive against thermal degradation, against UV light induced photochemical degradation, and during shelf life in cold storage, and that bioavailability of the vitamin in humans is at least as good as in a commercial food supplement based on synthetic surfactants11. We recently showed also that casein micelles with or without the use of calcium and phosphate, may encapsulate and protect omega 3 DHA12.
Cryo-TEM images (right) and Size distributions (left) of (A) naturally occurring CM in skim milk; (B) and (C) rCM; (D) D2-rCM. The bar on the bottom right is 100 nm long. (The dark area on the bottom is the perforated carbon film holding the sample.)10/
Proposed model for the re-assembly of CM, incorporating Vit D in their core11:
AFM analysis of rCM:
- Beta lactoglobulin-polysaccharide nanocomplexes as vehicles
We have introduced a novel nanodelivery technology for hydrophobic nutraceuticals, based on beta lactoglobulin- a molecular carrier of hydrophobic compounds, and an excess of pectin, which brought about the formation of stable electrostatic nanocomplexes useful for enrichment of clear acid beverages13, 14.
We were apparently the first to show that beta lactoglobulin can bind DHA, the important omega-3 fatty acid. The b-lg pectin nanocomplexes provided protection against DHA degradation during accelerated shelf life study15.
Left: b-lactoglobulin dimer structure. The b-barrel structure forms a solvent accessible cavity, which is a good binding site for certain hydrophobic ligands (like vitamins A, D) There are 3 possible binding sites (marked a-c). We have introduced a technology to nanoencapsulate hydrophobic nutraceuticals within b-Lg-pectin complexes, as modeled in the drawing on the right.
Model of the structure of the complexes as a function of pectin concentration.
- Heat denatured beta lactoglobulin as a vehicle for water-soluble bioactives
We introduced a novel technology for nanoencapsulation of EGCG- a highly potent polyphenolic nutraceutical with numerous attributed health benefits, which is water soluble (thus difficult to nanoencapsulate) within heat-denatured beta lactoglobulin nanoparticles. The nanoparticles formed provided good protection against EGCG degradation, and kept the system completely transparent. Moreover, the encapsulation significantly suppressed the bitter & astringent tastes of EGCG16-18. We have also obtained interesting insights on the protective effects of sugars against EGCG oxidative deterioration18.
Ranking provided by a sensory panel (n=6), evaluating bitterness and astringency of free EGCG compared to EGCG nanoparticles. The scale was: 1- not detectable, 2-faintly detectable, 3-detectable, 4-clearly detectable, 5 unpleasantly detectable. Significant differences (P<0.05) are designated with an asterisk
- Novel Maillard-reaction based protein-polysaccharide conjugates, as nanovehicles for hydrophobic nutraceuticals in clear beverages
We developed novel Maillard conjugates of milk proteins and oligosaccharides, and demonstrated the binding of hydrophobic nutraceuticals, and the protection conferred by the conjugate-based nanovehicles in clear solutions, applicable for nutraceuticals enrichment of clear beverages19.
Increased curvature effect by conjugating an oligosaccharide (MD) onto
the hydrophilic domains of casein.
- Biphasic co-assembly for creating novel functional nanostructures
We have managed to control the size of crystals of hydrophobic nutraceuticals and to nanoencapsulate them using hydrophobins- fungi based proteins, while providing significant protection to the hydrophobic nutraceutical against degradation. This is apparently first work showing the potential of hydrophobins for nanoencapsulation application in food20.
Crystals of hydrophobic nutraceuticals in aqueous environment. Left- Genistein (under polarized light), center- vitamin D, right – Naringenin.
Other amphiphilic proteins also exhibit the capability of controlling crystallization of hydrophobic bioactive compounds (see e.g. beta lactoglobulin effect on naringenin crystallization. The photograph shows naringenin in phosphate buffer solutions, without β-Lg (left) and with 0.54 mM β-Lg (right).)
- Novel arabinogalactan-based targeted delivery system for chemotherapy
We have managed to form (in collaboration with Prof. Avi Domb’s team, Hebrew University) a novel polymeric delivery system for anti-cancer drugs, based on arabinogalactan (a highly water soluble polysaccharide from the Larix tree). The system has both an active targeting mechanism, based on folic acid, and a target-activated release mechanism- by connecting the drug via an endosomally cleavable peptide linker, which remains intact in the circulation (see scheme below). We have successfully demonstrated the efficacy and selectivity of the system on cell lines21.
- Beta casein nanoparticles for oral delivery of anticancer drugs
We have harnessed the self-assembly of beta casein for nanoencapsulation of hydrophobic anticancer drugs, e.g. mitoxantrone, paclitaxel, irinotecan, docetaxel and vinblastine, and demonstrated release of paclitaxel for treatment of gastric carcinoma (one of the leading causes of death among cancer patients), upon simulated gastric digestion, and no cytotoxicity before digestion22-25.
A proposed model for the entrapment of hydrophobic drugs within beta casein micellar nanoparticles. Positively charged drugs may also bind to the negatively charged surface of the micelle, and induce micelle aggregation.
Solubilization of hydrophobic chemotherapeutic drugs by b-CN (left cuvettes) vs. pure drug in PBS at the same concentration as in β–CN solution (right cuvettes).
A model of b-CN-MX interactions and a TEM image supporting it26.
A model of b-CN-paclitaxel and b-CN-docetaxel nanoparticle formation22
Light microscope images of pure PTX vs. b-CN-PTX NPs in PBS24
CryoTEM Images of pure b-CN (left) and b-CN-PTX NPs in PBS showing PTX nanocrystal formed and covered with b-CN 24.
Cell cytotoxicity experiment on human gastric carcinoma cells, showing similar cytotoxicity of the released and the unencapsulated drug, vs no effect of the encapsulated drug 24
- Novel “Quadrugnostic” nanoparticles for combined diagnostics, drugs and chemosensitizers delivery
Anticancer drug resistance almost invariably emerges and poses major obstacles towards curative therapy of various human malignancies. We are developing innovative theragnostic nanovehicles which will harbor four major components: (1) a selective targeting moiety, (2) a diagnostic imaging aid for the localization of the malignant tumor and its metastases, (3) a cytotoxic, small molecule drug(s) or novel therapeutic biological(s), and (4) a chemosensitizer aimed at neutralizing a resistance mechanism of drug resistant cells. We proposed to name these envisioned four element-containing nanovehicle platform, “quadrugnostic” nanomedicine26. This targeted strategy holds promise in paving the way for the introduction of highly effective nanoscopic vehicles for cancer therapeutics while overcoming drug resistance.
A model of the quadrugnostic nanoparticle and its functions:
1. Livney, Y. D.; Ramon, O.; Kesselman, E.; Cogan, U.; Mizrahi, S.; Cohen, Y. Swelling of Dextran Gel and Osmotic Pressure of Soluble Dextran in the Presence of Salts Journal of Polymer Science, Part B: Polymer Physics 2001, 39, 2740-2750 http://onlinelibrary.wiley.com/doi/10.1002/polb.10038/full.
2. Livney, Y. D.; Portnaya, I.; Faupin, B.; Ramon, O.; Cohen, Y.; Cogan, U.; Mizrahi, S. Interactions between inorganic salts and polyacrylamide in aqueous solutions and gels Journal of Polymer Science Part B: Polymer Physics 2003, 41, 508-519 http://onlinelibrary.wiley.com/doi/10.1002/polb.10406/full.
3. Shechter, I.; Ramon, O.; Portnaya, I.; Paz, Y.; Livney, Y. D. Microcalorimetric Study of the Effects of a Chaotropic Salt, KSCN, on the Lower Critical Solution Temperature (LCST) of Aqueous Poly(N-isopropylacrylamide) (PNIPA) Solutions Macromolecules 2010, 43, (1), 480-487 http://pubs.acs.org/doi/abs/10.1021/ma9018312.
4. Livney, Y. D.; Portnaya, I.; Faupin, B.; Fahoum, L.; Ramon, O.; Cohen, Y.; Mizrahi, S.; Cogan, U. Interactions of glucose and polyacrylamide in solutions and gels Journal of Polymer Science Part B: Polymer Physics 2003, 41, (23), 3053-3063 http://onlinelibrary.wiley.com/doi/10.1002/polb.10632/full.
5. Shpigelman, A.; Paz, Y.; Ramon, O.; Livney, Y. Isomeric sugar effects on thermal phase transition of aqueous PNIPA solutions, probed by ATR-FTIR spectroscopy; insights to protein protection by sugars Colloid & Polymer Science 2011, 289, (3), 281-290 http://dx.doi.org/10.1007/s00396-010-2354-z.
6. Shpigelman, A.; Portnaya, I.; Ramon, O.; Livney, Y. D. Saccharide-structure effects on poly n-Isopropylacrylamide phase transition in aqueous media; reflections on protein stability Journal of Polymer Science Part B: Polymer Physics 2008, 46, 2307-2318 http://onlinelibrary.wiley.com/doi/10.1002/polb.21562/full.
7. Manukovsky, N.; Shpigelman, A.; Edelman, R.; Livney, Y. D. Hydration-mediated effects of saccharide stereochemistry on poly(N-isopropylacrylamide) gel swelling Journal of Polymer Science Part B: Polymer Physics 2011, 49, (7), 523-530 http://onlinelibrary.wiley.com/doi/10.1002/polb.22212/full.
8. Srebnik, S.; Matza, R.; Kusner, I.; Livney, Y. D. structural effect of sugars on water, American Physical Society (APS) Meeting March 2009, Pittsburg, Pennsylvania, USA, 2009; Pittsburg, Pennsylvania, USA, 2009.
9. Livney, Y. D.; Edelman, R.; Kusner, I.; Kisiliak, R.; Srebnik, S. Water-structure effect of sugar stereochemistry, and its impact on protein thermal stability Frontiers in Water Biophysics, Trieste, Italy, May 23-26, 2010; Trieste, Italy, 2010; http://waterbiophysics.eu/en/upload/2012/Book%20TRIESTE%20WATER%20-FINAL%20WEB.pdf.
10. Semo, E.; Kesselman, E.; Danino, D.; Livney, Y. D. Casein micelle as a natural nano-capsular vehicle for nutraceuticals Food Hydrocolloids 2007, 21, (5-6), 936-942 http://www.sciencedirect.com/science/article/pii/S0268005X06002098.
11. Haham, M.; Ish-Shalom, S.; Nodelman, M.; Duek, I.; Segal, E.; Kustanovich, M.; Livney, Y. D. Stability and bioavailability of vitamin D nanoencapsulated in casein micelles Food & Function 2012, 3, (7), 737-744 http://pubs.rsc.org/en/content/articlehtml/2012/fo/c2fo10249h.
12. Zimet, P.; Rosenberg, D.; Livney, Y. D. Re-assembled casein micelles and casein nanoparticles as nano-vehicles for [omega]-3 polyunsaturated fatty acids Food Hydrocolloids 2011, 25, (5), 1270-1276 http://www.sciencedirect.com/science/article/B6VP9-51MDSJ2-7/2/4a49e7e36bbd0cabe3704d268812cfc6.
13. Ron, N.; Zimet, P.; Bargarum, J.; Livney, Y. D. Beta-lactoglobulin-polysaccharide complexes as nanovehicles for hydrophobic nutraceuticals in non-fat foods and clear beverages International Dairy Journal 2010, 20, (10), 686-693 http://www.sciencedirect.com/science/article/B6T7C-5051PFV-1/2/aeacf364f21ac89dfc3b1a728f861b73.
14. Ron, N. Beta-lactoglobulin as a Nano-capsular Vehicle for Hydrophobic Nutraceuticals. M.Sc., The Technion, Israel Institute of Technology, Haifa, Israel, 2007.
15. Zimet, P.; Livney, Y. D. Beta-lactoglobulin and its nanocomplexes with pectin as vehicles for omega-3 polyunsaturated fatty acids Food Hydrocolloids 2009, 23, (4), 1120-1126 http://www.sciencedirect.com/science/article/pii/S0268005X08002476.
16. Shpigelman, A.; Israeli, G.; Livney, Y. D. Thermally-induced protein-polyphenol co-assemblies: beta lactoglobulin-based nanocomplexes as protective nanovehicles for EGCG Food Hydrocolloids 2010, 24, (8), 735-743 http://www.sciencedirect.com/science/article/pii/S0268005X10000615.
17. Shpigelman, A.; Cohen, Y.; Livney, Y. D. Thermally-induced β-lactoglobulin–EGCG nanovehicles: Loading, stability, sensory and digestive-release study Food Hydrocolloids 2012, 29, (1), 57-67 http://www.sciencedirect.com/science/article/pii/S0268005X12000288.
18. Shpigelman, A.; Zisapel, A.; Cohen, Y.; Livney, Y. D. Mechanisms of saccharide protection against epigallocatechin-3-gallate deterioration in aqueous solutions Food Chemistry 2013, 139, (1–4), 1105-1112 http://www.sciencedirect.com/science/article/pii/S0308814613000447.
19. Markman, G.; Livney, Y. D. Maillard-conjugate based core–shell co-assemblies for nanoencapsulation of hydrophobic nutraceuticals in clear beverages Food & Function 2012, 3, 262-270 http://pubs.rsc.org/en/content/articlehtml/2012/fo/c1fo10220f.
20. Israeli-Lev, G.; Livney, Y. D. Self-assembly of hydrophobin and its co-assembly with hydrophobic nutraceuticals in aqueous solutions: Towards application as delivery systems Food Hydrocolloids 2014, 35, 28-35 http://www.sciencedirect.com/science/article/pii/S0268005X13002282.
21. Pinhassi, R. I.; Assaraf, Y. G.; Farber, S.; Stark, M.; Ickowicz, D.; Drori, S.; Domb, A. J.; Livney, Y. D. Arabinogalactan−Folic Acid−Drug Conjugate for Targeted Delivery and Target-Activated Release of Anticancer Drugs to Folate Receptor-Overexpressing Cells Biomacromolecules 2010, 11, (1), 294-303 http://dx.doi.org/10.1021/bm900853z.
22. Shapira, A.; Assaraf, Y. G.; Epstein, D.; Livney, Y. D. Beta-casein Nanoparticles as an Oral Delivery System for Chemotherapeutic Drugs: Impact of Drug Structure and Properties on Co-assembly Pharmaceutical Research 2010, 27, 2175–2186 http://link.springer.com/article/10.1007/s11095-010-0222-7.
23. Shapira, A.; Assaraf, Y. G.; Livney, Y. D. Beta-casein nanovehicles for oral delivery of chemotherapeutic drugs Nanomedicine: Nanotechnology, Biology, and Medicine 2010, 6, 119-126 http://www.sciencedirect.com/science/article/pii/S1549963409001130.
24. Shapira, A.; Davidson, I.; Avni, N.; Assaraf, Y. G.; Livney, Y. D. β-Casein nanoparticle-based oral drug delivery system for potential treatment of gastric carcinoma: Stability, target-activated release and cytotoxicity European Journal of Pharmaceutics and Biopharmaceutics 2012, 80, (2), 298-305 http://www.sciencedirect.com/science/article/pii/S0939641111003171.
25. Shapira, A.; Markman, G.; Assaraf, Y. G.; Livney, Y. D. β-casein–based nanovehicles for oral delivery of chemotherapeutic drugs: drug-protein interactions and mitoxantrone loading capacity Nanomedicine: Nanotechnology, Biology and Medicine 2010, 6, (4), 547-555 http://www.sciencedirect.com/science/article/pii/S1549963410000109.
26. Shapira, A.; Livney, Y. D.; Broxterman, H. J.; Assaraf, Y. G. Nanomedicine for targeted cancer therapy: Towards the overcoming of drug resistance Drug Resistance Updates 2011, 14, (3), 150-163 http://www.sciencedirect.com/science/article/pii/S1368764611000045.