Technical field

Biomedical application , nanotechnology Background Art

Hydroxyapatite (HAp) is the molecular formula of which Ca ₁₀ (PO4)6(OH) ₂ is a bioactive ceramic with a crystal structure similar to native bone and teeth minerals due to its structural similarity to bone mineral, enamel and dentin. HAp ceramics have gained acceptance as bone substitute, materials in dentistry and medicine due to its ability to form bone apatite-like and has the ability to develop a direct adherent and strong bonding with the bone tissue. Moreover, it provides a scaffold or template for the formation of new bone. The applications of HAp as biomaterial are: repair of bony and periodontal defects, maintenance or augmentation of alveolar ridge, ear implant, spine fusion, adjuvant to uncoated implants, maxillofacial surgery. HAp has shown strong inhibitory effects on the growth of various cancers with minimal side effects, and its nanoparticles have stronger anti-tumor effect than macromolecules. Also, it could bring strong cooperative effect with chemotherapy medicine which, in turn, will result in reducing the toxicity of it.

There has been an increasing concern over the discharge of heavy metals into the environment and there are several methods for heavy metal removal from wastewaters. In particular, chemical stabilization is one of the methods of reducing the leachability of heavy metals in waste materials, with forming new, less soluble mineral phases that are more stable in the environment. Hydroxyapatite is an ideal material for long- term containment of contaminants It is well documented that HAp strongly sorbs uranium and many other radionuclides and heavy metals. HAp has been investigated as a potential agent to treat heavy metal- contaminated soils, sediments, wastes, and wastewater, especially in the cases of Pb, Cd, Zn, and U. HAp is an economical and natural source of phosphate, which has a high removal capacity for divalent heavy metal ions. Although several researches achieve nano-hydroxyapatite but the tested one for intravenous injection has large particles. It was difficult to inject hydroxyapatite intravenously because the particles of this material which previously prepared were lager enough to aggregate and form secondary larger particles. These formed particles cause blockage of capillaries and leading to death. For this reason, hydroxyapatite material did not use as intravenous therapy. Until the date of issue, There is no previous work studying the effect of this material on DNA or the effect of it against heavy metal or toxic substance in vivo.

Disclosure of Invention :

Nano-hydroxyapatite had been synthesized by an organic-inorganic complexion route. Such rout succeeded in synthesis nano-hydroxyapatite in very fine spherical particles in nanosize. The advantages of this technique showed to have: low processing temperature, high molecular level homogeneity, improved purity, morphology, texture, and a scope to tailor the made compound. The surface area of the formed nano HAp was about 6 times larger than the surface area of HAp prepared in another studies which was used in intravenous injection of male Wistar rats. The transmission electron microscope showed a nano-HAp of ultra small crystals distributed in matrix as shown in figure 1 with average grain size of 40 nm. The surface area of the dried powder was 405m /g with total pore diameter 0.4621 cc/g. the average pore diameter was 45.63 A.

Tested animals were intraperitonoully injected with LD ₅₀ of toxic heavy metal after three hours the synthesized nano-hydroxyapatite was injected intravenously. The analysis results of DNA in Blood and liver samples which were collected through 48hours showed in figure 2 & 3 respectively a succeeding of nano-hydroxyapatite in repairing the fragmented DNA.

Also, heart, liver and thyroid function enzymes were evaluated as markers of general health and the results confirmed the bio-safe usage of this synthesized material. The same experiment was repeated with another concentration of nano-hydroxyapatite and the results were the same for three years and all the tested animals treated still alive with a good health until the date of issue.

The prepared nano-hydroxyapatite succeeded in repairing the fragmented DNA of the rats which had been exposed to LD ₅₀ of toxic heavy metal within 48 hours after intravenous injection with single dose only and the tested animals were alive without any side effects.

Also, heart, liver and thyroid function enzymes were evaluated as markers of general health and the results confirmed the bio-safe usage of this synthesized material. The same experiment was repeated with another concentration of nano-hydroxyapatite and the results were the same for three years.

Therefore, the prepared nano-hydroxyapatite can be used safely as a thereby by intravenous injection in suitable doses for protection against contamination of heavy metal and radioactive materials and for recovery from different diseases such as cancer and osteoporosis.

Brief Description of Drawings:

Figure 1 TEM migrograph analysis of nano-HAp with its the diffraction pattern.

Figure 2 Blood DNA electrophoresis of tested animals where a)control animal

b&c) toxic heavy metals treated animals, d&e) nano-HAp after toxic heavy metal treatment

Figure 3 Blood DNA electrophoresis of tested animals where

a) Control animal group b) Toxic heavy metal treated animals c) Toxic heavy metal treated animals d &e ) Intravenous of low concentration of nano-HAp after toxic heavy metal treatment f&g) intravenous of high concentration of nano- HAp after toxic heavy metal treatment

Figure 4 comet of DNA in blood

a) Control animal group b) Toxic heavy metal treated animals c) Toxic heavy metal treated animals d &e ) Intravenous of low concentration of nano-HAp after toxic heavy metal treatment f&g) intravenous of high concentration of nano- HAp after toxic heavy metal treatment

Figure 5 liver DNA electrophoresis of tested animals where

a) Control animal group b) Toxic heavy metal treated animals c) Toxic heavy metal treated animals d &e ) Intravenous of low concentration of nano-HAp after toxic heavy metal treatment f&g) intravenous of high concentration of nano- HAp after toxic heavy metal treatment

Figure 6 comet of DNA in liver

a) Control animal group b) Toxic heavy metal treated animals c) Toxic heavy metal treated animals d &e ) Intravenous of low concentration of nano-HAp after toxic heavy metal treatment f&g) intravenous of high concentration of nano- HAp after toxic heavy metal treatment Best Mode for carrying out the invention :

The synthesized nano-hydroxyapatite succeeded in repairing the fragmented DNA of the rats which had been exposed to LD ₅₀ of toxic heavy metal within 48 hours after intravenous injection with single dose only and the tested animals were alive without any side effects.

Therefore the synthesized nano-hydroxyapatite could be injected intravenously safely in:

1 - Repair fragmented DNA in blood and liver.

2- protection against internal contamination of heavy metal and radioactive materials which cause fragmentation of DNA.

3- different concentration suitable for treatment of many diseases, such as cancer and osteoporosis.