Toxicological Impacts:Nutrition and Nanotechnology

Bernadene Magnuson, Ph.D.

Uses and benefits• Improved uptake of low bioavailability

nutrients or bioactive food compounds• Enhance uptake of nutrients in individuals

with absorption disorders• Alter hydrophobicity/lipophilicity of

nutritional fortifications• Improved stability and sensory qualities of

fortified food

Key Points

1. Nutritional products claiming to usenanotechnology are available in the market.

2. Nanotechnology can be used to alter bioavailability of nutrients and bioactives.

3. The potential toxicity of nutrientscan be affected by a changein particle size.

Examples of productsA Nanotechnology Consumer Products Inventory

Displaying records 1-10 of 64 for “food”Includes packaging, appliances, supplements

June 20, 2007http://www.nanotechproject.org/44

http://www.nutralease.com/products.aspNutraleaseCo-enzyme Q10 (Ubiquinon)LuteinLycopenePhytosterol (sitosterol)Vitamin DVitamin E

•patent pending Nano-sized Self-assembled Liquid Structures (NSSL)•vehicles are expanded micelles (~30 nm); fortifying nano-vehicles ( FNVs).

Canola Active •“Nanocapsules in cooking oil to improve bioavailability of nutraceuticals, for example, plant sterols to reduce the body’sabsorption of cholesterol in the blood.”

•Incorporates phytosterol-filled nano-vesicles from Nutralease into canola oil

•On the market in Israel

•Shemen Industries Ltd

Aquanova, Germany• Use nanotechnology to

produce micelles – to improve solubility of

insoluble bioactives – to change water/fat solubility

of nutrients– Vitamins A, C, D, E, K– Coenzyme 10– β-carotene,– isoflavones,– α-lipoic acid,– omega fatty acids

http://www.aquanova.de/

Nano-tea

Nano-Green Tea•• Nano-Dark-Green Tea• Nano-White Tea• Nano-Black Tea• Nano-Yellow Tea• Nano-Dark Tea• Nano-Selenium Rich White Tea• Nano-Selenium Rich Dark Green Tea• Nano-Selenium Rich Black Tea• Nano-Selenium Rich Green Tea• Nano-Selenium Rich Yellow Tea • Nano-Selenium Rich Dark Tea

http://www.369.com.cn/En/default.htm

NanoGreensIngredient list•Greens Blend (Proprietary) 2350 mg

Barley Grass Juice Powder*, Spirulina*, Chlorella (Japanese soft shell)• Phyto-Nutrient Blend (Proprietary) 325 mg

Blueberry, Green Tea Extract, Grape Seed Extract, Cranberry, Raspberry, Tart Cherry, Pine Bark Extract, Broccoli, Tomato, Carrot, Spinach, Kale, Brussels Sprout, Bilberry, Elderberry,Pomegranate, Blackberry

• Isoquercitin/Rutin 50/50 160 mg• Raspberry Extract (20% Ellagic Acid) 50 mg• Fruit & Vegetable Blend 900 mg

•Apple*, Carrot*, Mango*, Sweet Potato*, Lemon*, Parsley*, Peach*, Kale*, Broccoli*, Spinach*, Leek*, Beet* , Cranberry* (Quinic Acid 6%)

• Acerola Cherry Powder* (17.5% Ascorbic Acid) 175 mg• Rice Bran Soluble* 2500 mg• Aloe Vera Powder Extract* (100:1 freeze dried) 30 mg• Green Tea, White Tea (decaffeinated, 50% Polyphenol) 100 mg• Polygonum Cuspidatum (15% Resveratrol) 50 mg• Oat Beta Glucan* 2200 mg• Cinnamon Blend (Proprietary) 50 mg (Cinnamon Extract 8%, Cinnamon Bark Powder)• Milk Thistle (20% Silymarin) 50 mg• Marigold Extract (5% Lutein with Zeaxanthin) 50 mg• Dunaliella Salina (Natural Carotenoids) 100 mg• Enzymes (plant-based) 40 mg

•Alpha Amylase, Bromelain, Cellulase, Galactosidase, Glucoamylase, Hemicellulase, Lipase, Papain, Protease

• Lecithin (non GMO) 1925 mg• Cabbage (Japanese, fermented) 30 mg• Lycopene Extract-10% (from tomato) 25 mg• Lemon Peel Powder* 25 mg• Quinoa Sprout* 90 mg• Artichoke Extract (5% Cynarin) 20 mg• Atlantic Kelp Powder* (Laminara Digitata) 20 mg•Natural flavors (plant-based), stevia, NanoSorb (phospholipids, lipid esters), citric acid.

“NanoGreens10 is formulated with patented "NanoSorb"ô which utilizes nanosized vesicles that spontaneously encapsulate nutraceuticals to maximize availability for absorption by the small intestine!”

Ellagic acid

Quercitin

http://www.biopharmasci.com

General concerns over nanoscale versus microscale materials

• Higher exposure per unit mass – Small size, large surface area, may result in increased ability to

generate Reactive Oxygen Species (ROS)• Routes of exposure may differ due to smaller size.

– E.g. olfactory transport, dermal penetration• Different distribution to tissues by virtue of their different

size or surface coating/chemistry.– E.g. Inflammatory responses induced by fine and ultrafine TiO2

• “Novel property” of nanoscale materialmay translate into a new mode of action.

Nel et al., Science 2006

Factors affecting uptake and translocation of orally administered nanoparticles

• Diameter • below 1µm, decreased diameter increased uptake

• Surface charge • non-ionic uptake higher

• Shape and elasticity • no clear shape effect, elasticity increases ability to traverse

capillaries

• Physical and chemical stability• Colloidal instability leads to aggregation, chemical stability

affects biodegradability and release of encapsulated material

Florence, Drug Disc Today 2005

Issues regarding nanoparticles for nutrient delivery

• Safety of the nanoparticle delivery system per se

One approach

Delivery vehicle not absorbed- Nutralease, Aquanova

http://www.nutralease.com/products.asp

Toxicity evaluation of “void” nanoparticle•Polymeric particles : NIPAA-M/VP/PEG-A• N-isopropylacrylamide/N-vinyl-2pyrrolidone/Polyethylene glycol monoacrylate

•Cytotoxicity to various cancer cell lines

•Administration to mice twice a week for 3 weeks•No change in body weight•No gross pathology on necropsy

J. Nanobiotechnology, 2007

Issues regarding nanoparticles for nutrient delivery

• Safety of the nanoparticles per se• Change in toxicity of nutrient/bioactives

due to increased uptake or altered distribution in body– Nutrients with known toxicities– Compounds that have low bioavailability

• Impact on nutritional labeling? – how calculate %DV if change bioavailability?

Bioavailability of nano-nutrients

• Water-soluble vitamin E (Aquanova) – Back et al., 2007

• Ferric phosphate nanoparticles– Rohner et al., 2007

• Vitamin E (PEG encapsulated) nanospheres – Shea et al., 2005

• Selenium - Zhang et al., 2001

• Copper - Chen et al., 2006

• Zinc - Wang et al., 2006

Nanoparticles in the intestine

Water-soluble α-tocopherol

•Bioavailability of water-soluble α-tocopherol (100 IU) was ~50% greater than bioavailability of a commercial fat-soluble α-tocopherol (100 IU) preparation in 14 human subjects given a single oral dose.

Back et al., Eur J. Nutr. 2007

Bioavailability of ferric phosphate nanoparticles in rats

• Background– Highly bioavailable water-soluble FeSO4

supplements cause adverse organoleptic changes in foods

– Low solubility Fe compounds, more stable in foods, less organoleptic change, but poor bioavailability

• Objective: improve bioavailability of FePO4by decreasing particle size to nm

Rohner et al., 2007

Synthesis of FePO4 nanoparticlesFlame spray pyrolysis used to produce particles varying in diameter and specific surface area

A. Large – 64.2 nm diam, 32.6 m2/g SSAB. Medium – 30.5 nm diam, 68.6 m2/g SSAC. Small – 10.7 nm diam, 194.7 m2/g SSA

In vitro solubility test of the 3 FePO4 compounds and FeSO4 at pH 1

Copyright ©2007 American Society for Nutrition

Dose-response curves: Hb repletion assay in Fe-depleted rats consuming a Fe-def diet or the Fe-def diet fortified with

FeSO4, FePO4 small, FePO4 medium, or FePO4 large particles for 15 days

Toxicity of FePO4 nanoparticles• Evaluated histology

– liver, spleen, kidney, stomach, duodenum, jejunum, ileum, colon, pancreas, lymphatic tissue and sternum

• Special stains for Fe deposits• TEM for damage to duodenum mucosa• Plasma TBARS for reactive oxygen• No evidence of potential toxicity in Fe-

depleted rats fed FePO4 nanoparticles for 15 days

Summary of findings

• Nanotechnology can be used to alter form of nutrients such that solubility properties are altered, but physiological properties retained

• Nanotechnology can improve bioavailability of poorly absorbed nutrients

• May be beneficial in cases of malabsorption and for improved fortification

Acute toxicity of copper micro-and nanoparticles

• Background– Copper is essential micronutrient– Copper toxicity results in hemolysis and liver

and kidney damage• Objective

– Compare acute toxicity of oral administration of copper micro- and nano-particles

Chen et al., 2006 Tox Lett

Test materials• Nano-copper

– average size 23.5 nm (distribution not given);– SSA 2.95 x 105 cm2/g– Particle number 1.7 x1010 /µg

• Micro-copper– average size 17µm (distribution not given);– SSA 3.99 x 102 cm2/g– Particle number 44/µg

• Mice- ICR strain, both sexes, age 8 wks,- single oral gavage, tissues collected 48 h after dosing

Chen et al., 2006 Tox Lett

Results

Group BUN(mmol/L)

Cr(µmol/L)

ALP(IU)

LD50(mg/kg)

MicroCu700 mg/kg

8.0 49.0 92 >5000

NanoCu700 mg/kg

14.3* 66.0* 186* 413

ControlVehicle only

8.8 51.8 112 NA

Renal function tests: BUN= Blood urea nitrogen, Cr= Creatinine ,Liver damage: ALP=alkaline phosphatase* P<0.05 vs control

Chen et al., 2006 Tox Lett

Pathological changes in kidney

Chen et al., 2006 Tox Lett

Pathological changes in kidney

Control (a); lower dose group N1 (b); medium dose group N4 (c) and higher dose group N7 (d). A: renal glomerulus and B: Bowman's capsule. Magnification = 100X

Pathological changes in the spleen

Chen et al., 2006 Tox Lett

Pathological changes in the spleen

Control (a); groups N1 (b); N4 (c) and N7 (d). A: splenic unit and B: lymphocytes. Magnification = 40X

Summary• Data do not imply toxicity due to nanoparticle per

se, no evidence of uptake of particle• The difference in size of Cu particles altered

biological effects, likely due to release of Cu ions• Comparison of toxicity/biological activity based

on SSA (700 fold diff) rather than mg/kg may be more appropriate

• Demonstrates that cannot assume that non-toxic compounds will remain non-toxic

• If there is a change in physical properties need to determine if changes biological effects.

Conclusions

1. Nutritional products claiming to usenanotechnology are available in the market.

2. Nanotechnology can be used to alter bioavailability of nutrients and bioactives.

3. The potential toxicity of nutrientscan be affected by a changein bioavailability and/orparticle size.

Key References

• Back EI et al., 2006. Eur J Nutr 45(1):1-6• Bisht S et al., 2007. J Nanobiotech 17(5):3• Chen Z et al., 2006 Tox Lett 163:109-120• Florence AT 2005. Drug Disc Today 2:75• Nel A et al., 2006. Science 311:622-627• Rohner F et al., 2007. J Nutr 137:614-619• Shea TB et al., 2005. J Alzh Dis 7:297-301