Patent Application
20060246096
The present invention relates to core-shell polymers of poly-γ-glutamic acid (PGA). The core is prepared of cross-linked poly-γ-glutamic acid compounds, that are prepared by amidizing PGA with diamino compounds. The core and the shell could be hydrophilic, hydrophobic or amphiphilic.
The starting material of the present invention is PGA which is prepared by fermentation with a suitable microorganism, capable of producing PGA in a suitable fermentation medium, under conditions and time appropriate for the microorganism used.
The resulting culture medium is treated, by centrifugation, to separate the cells from the PGA. The resulting cell-free liquid is treated with acetone to obtain the PGA from this fermentation medium.
After this, the obtained PGA was purified by dialysis and freeze dried.
The molecular weight of the PGA is about 1,000,000.
The PGA is then partially amidated by reaction with a diamino compound. The diamino compound could be:
NH2—CH2—CH2—(O—CH2—CH2)n-NH2 n=2 to 12
EDBEA (2,2′-(Ethylenedioxy)bis(ethylamine)
This reaction determines the cross-linking of PGA. This cross-linking was performed in different proportions, from 1 to 99%.
This reaction takes place in water, in the presence of a water soluble carbodiimide (CDI) (eg., 1-[3-(dimethylamino)propyl]-3-Ethylcarbodiimide hydrochloride, etc).
The core is formed by crosslinking the PGA. In the second stage, the outer shell is formed from hydrophilic or hydrophobic polymer that optionally may be cross-linked a priori. The core-shell morphology may be a result of self assemble of modified PGA in the case when hydrophobic side chains ate introduced. Therefore, in aqueous solution the hydrophobic 0 chains are in the inner part and the residual hydrophilic segments occupy the corona. These particles are design for solubilization of water insoluble compounds or drugs.
The core and the outer shell can be formed independently to be hydrophobic or hydrophilic. The reaction takes place in a multi-step process. The formation of the primary crosslinked core is followed by formation of a covalently attached shell. The reaction is controlled by the conditions of reaction (e.g., concentration) to obtain either a very slightly crosslinked core wherein the porosity is very high, or to obtain a very highly crosslinked core wherein the porosity is very low. The degree of cross-linking could be in the range of from about 1% to about 99%.
In the present invention the core shell polymers of poly-γ-glutamic acid can be formed from the following ingredients:
In a preferred embodiment the range can be
A more preferred embodiment has the following composition:
The pH may be in the range of 6.8 to 7.8
The core polymer may be formed by taking a quantity of the PGA formed in the manner described above in water, adding 0.01 to about 0.7 CDI and stirring for a suitable period of time. To the resulting solution a diamine compound is added. The diamino may be present in a range of 5 to 400 μl. In-a preferred embodiment about 10-250 μl diamine may be added depending on the diamine selected.
Preparation of Poly-γ-Glutamic Acid
A solution was prepared by dissolving the following ingredients in 3 liters of distilled water.
The pH was adjusted to 7.4 with NaOH. The medium was autoclaved.
The Bacillus licheniformis suspension was used to inoculate the flasks which contain the medium solution, and they were incubated on the shaker (150 rpm) for seven days, at 37 C. The contents of the culture flasks were centrifuged to separate the cells from the polymer solution. Two volumes of 99.5% acetone were added slowly to the supernatant liquid while stirring. The liquid was decanted and the precipitated polymer was dissolved in distilled water. The resulting polymer solution was dialyzed 1 day against EDTA solution, and 6 days against distilled water and freeze dried.
Preparation of hydrophilic core polymer (10% of the free carboxyl groups are reacting) To a 10 g/l of 0.2 g of the PGA from Example 1 in water, 0.0433. CDI was added, and stirred 30 minutes. To the resulting solution 11.32 μl EDBEA was added, and stirred at ambient temperature for 24 hours. After this time, the resulting polymer solution was dialysed 7 days against distilled water and freeze dried.
Preparation of hydrophilic core polymer (50% of the free carboxyl groups are reacting) To a 10 g/l of 0.2 g of the PGA from Example 1 in water, 0.2164 CDI was added, and stirred 30 minutes. To the resulting solution 56.6 μl EDBEA was added, and stirred at ambient. temperature for 24 hours. After this time the resulting polymer solution was dialysed 7 days against distilled water and freeze dried.
Preparation of hydrophobic core polymer (50% of the free carboxyl groups are reacting)
To a 10 g/l of 0.5 g of the PGA from Example 1 in water, 0.5391 g CDI was added, and stirred 30 minutes. To the resulting solution 191.2 μl butylamine was added and stirred at ambient temperature for 24 hours. After this time the resulting polymer solution was dialysed 7 days against distilled water and freeze dried.
Preparation of hydrophobic core polymer (50% of the free carboxyl groups are reacting) To a 10 g/l of 0.5 g of the PGA from Example 1 in water, 0.5391 g CDI was added and stirred 30 minutes. To the resulting solution 211.5 μl benzylamine was added, and stirred at ambient temperature for 24 hours. After this time, the resulting polymer solution was dialysed 7 days against distilled water and freeze dried.
This is a conversion of provisional patent application Ser. No. 60/532,989 filed Dec. 29, 2003 the disclosures of which are incorporated by reference. U.S. Patent Documents U.S. Pat. No. 5,298,559 * Fujii et al. U.S. Pat. No. 5,324,780 * Oshima et al. U.S. Pat. No. 5,451,641 * Eisenhart et al. U.S. Pat. No. 5,453,458 * Takeuchi et al. U.S. Pat. No. 6,165,563 * Chandran et al. U.S. Pat. No. 6,326,511 * Borbely J.
| Number | Date | Country | |
|---|---|---|---|
| 60532989 | Dec 2003 | US |
