5 g of chitosan ( mw = 215 , 000 g / mol , deacetylation degree = 80 - 90 %, adrich - sigma ) was suspended in 2 - propanol ( 50 ml ) at room temperature while being stirred for 30 min . the resulting suspension was gently mixed with 12 . 5 ml of naoh solution to obtain a mixture , and the substitution degree of hydrophilic group can be adjusted by the concentration of naoh in the mixture . herein , the mixture contained 13 . 3 m naoh . then , the mixture was reacted with chloroacetic acid , followed by drying , to obtain soluble carboxymethyl - modified chitosan sample . synthesis of chitosan derivatives with hydrophobic hexanoyl modification and hydrophilic carboxymethyl modification 2g of dried chitosan derivatives with hydrophilic carboxymethyl modification prepared in comparative example 1 was dissolved in distilled water ( 50 ml ) while being stirred for 24 hours . then , the resulting solution was mixed with methanol ( 50 ml ), followed by the addition of hexanoyl anhydride at concentration of 0 . 2 m to obtain a reaction solution . after reacting for 20 hours , the reaction solution was collected by dialysis with ethanol solution ( 25 % v / v ) for 24 hours . after dying the resulting solution , chitosan derivatives with hydrophobic hexanoyl modification and hydrophilic carboxymethyl modification , called amphiphilic chitosan derivatives , were obtained , which can be represented by the following formula ( ii ). in addition , the sites of the substitution groups and the degree of hexanoyl substitution were confirmed by 1 h nmr and elemental analysis for n content . in the present embodiment , the degree of hexanoyl substitution of the chitosan derivatives is 13 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 1 , except that the concentration of the hexanoyl anhydride is 0 . 4 m . the degree of hexanoyl substitution of the chitosan derivatives prepared in the present embodiment is 26 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 1 , except that the concentration of the hexanoyl anhydride is 0 . 5 m . the degree of hexanoyl substitution of the chitosan derivatives prepared in the present embodiment is 33 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 1 , except that the concentration of the hexanoyl anhydride is 1 . 0 m . the degree of hexanoyl substitution of the chitosan derivatives prepared in the present embodiment is 48 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 1 , except that hexanol anhydride is substituted with decanoic anhydride . the degree of decanoyl substitution of the chitosan derivatives prepared in the present embodiment is 12 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 2 , except that hexanol anhydride is substituted with decanoic anhydride . the degree of decanoyl substitution of the chitosan derivatives prepared in the present embodiment is 24 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 3 , except that hexanol anhydride is substituted with decanoic anhydride . the degree of decanoyl substitution of the chitosan derivatives prepared in the present embodiment is 34 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 4 , except that hexanol anhydride is substituted with decanoic anhydride . the degree of decanoyl substitution of the chitosan derivatives prepared in the present embodiment is 45 %. 2g of dried chitosan derivatives with hydrophilic carboxymethyl modification prepared in comparative example 1 was dissolved in distilled water ( 50 ml ) while being stirred for 24 hours . then , the resulting solution was mixed with methanol ( 50 ml ), followed by the addition of dodecanoic anhydride at concentration of 0 . 2 m to obtain a reaction solution . the reaction solution was reacted under 50 ° c . for 20 hours , followed by being reacted at room temperature for 18 hours , and then the reaction solution was collected by dialysis with ethanol solution ( 25 % v / v ) for 24 hours . after dying the resulting solution , a chitosan derivatives with hydrophobic dodecanoyl modification and hydrophilic carboxymethyl modification , called amphiphilic chitosan derivatives were obtained . in addition , the sites of the substitution groups and the degree of dodecanoyl substitution were confirmed by 1 h nmr and elemental analysis for n content . in the present embodiment , the degree of dodecanoyl substitution of the chitosan derivatives is 10 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 9 , except that the concentration of the dodecanoic anhydride is 0 . 4 m . the degree of dodecanoyl substitution of the chitosan derivatives prepared in the present embodiment is 22 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 9 , except that the concentration of the dodecanoic anhydride is 0 . 5 m . the degree of dodecanoyl substitution of the chitosan derivatives prepared in the present embodiment is 30 %. the reaction condition and steps in the present embodiment are the same as those in embodiment 9 , except that the concentration of the dodecanoic anhydride is 1 . 0 m . the degree of dodecanoyl substitution of the chitosan derivatives prepared in the present embodiment is 44 %. 100 mg / ml of chitosan derivatives prepared by comparative example 1 and embodiments 1 - 12 was suspended in a distilled water under gentle shaking at room temperature for 24 hours , followed by ultrasonication using a probe type sonifier ( automatic ultrasonic processor uh - 500a ) for 2 min . after repeating the sonication three times , the final products were obtained . in order to identify whether the products were in a form of sphere , transmission electron microscopy ( tem ) ( jeol2100 , japan ) was used to check the shape of the products and determine the size of the products . according to tem image , it can be found that the amphiphilic chitosan derivatives can self - assemble to form hollow spheres with nano or micro sizes , through the interaction between the hydrophobic groups , wherein the hollow spheres have a double layer structure , i . e . hydrophilic - hydrophobic - hydrophilic configuration . the mean size and size distribution of the hollow spheres from the amphiphilic chitosan derivatives prepared by comparative example 1 and embodiments 1 - 4 were measured by dynamic light scattering ( dls ) nanoparticle size analyzer ( lb - 550 , horiba , japan ). in addition , the critical aggregation concentration ( cac ) of the amphiphilic chitosan derivatives was obtained from the fluorescent spectrum , wherein pyrene was used as a fluorescence probe due to its high affinity to hydrophobic micro - domains . the smaller of the critical aggregation concentration means the easier of forming hollow spheres . the characters of the mean size and the critical aggregation concentration of the hollow spheres from the amphiphilic chitosan derivatives are shown in table 1 . from the results shown in table 1 , the amphiphilic chitosan derivatives can form two kinds of hollow spheres with different mean sizes . as the substitution degree of hydrophobic group increases , the amphiphilic chitosan derivatives tend to form hollow spheres with mean size 2 . fig1 shows the relation between the substitution degree of hydrophobic group and mean size distribution . as shown in fig1 , most of the amphiphilic chitosan derivatives prepared in embodiments 3 and 4 form hollow spheres with a particle size of 200 nm . measurement of the thickness of the shells of the hollow spheres from the amphiphilic chitosan derivatives in order to confirm the amphiphilic chitosan derivatives forming hollow spheres , the hollow spheres from the amphiphilic chitosan derivatives were dehydrated to remove water inside and outside the hollow spheres . then , scanning electron - microscopy ( sem ) ( s6500 , jeol , japan ) was used for detecting the dehydrated hollow spheres from amphiphilic chitosan derivatives . sem image shows that the hollow spheres with particle size of 200 nm each lost their structural integrity , and collapsed to resemble a flattened basketball after dehydration . furthermore , the thickness of the double shell is about 9 nm , which can be found from sem image , so the thickness of the single shell is calculated to be about 4 . 5 nm . measurement of the encapsulation efficiency of the hollow sphere from amphiphilic chitosan derivatives doxorubicin ( dox ) was provided and mixed with 20 ml of chitosan derivatives solution prepared by using comparative example 1 and embodiments 1 - 12 , respectively . the final concentration of the dox in the chitosan derivatives solution is 20 μg / ml . after stirring for 24 hours , the resulting solution was ultrasonicated with sonicator ( automatic ultrasonic processor uh - 500a ), and the chitosan derivatives self - assembled to form hollow spheres , wherein the dox was encapsulated inside the hollow spheres to form an amphiphilic chitosan derivative complex for medical use . insoluble and free dox was removed by centrifugation at 2000 rmp under 4 ° c . for 5 mins . then , the amphiphilic chitosan derivative complex for medical use was separated from the solution by centrifugation at 15000 rmp under 4 ° c . for 15 mins . free dox concentration in the supernatant was analyzed by uv - vis spectrometer ( sp - 8001 , metertech inc .). encapsulation efficiency ( ee ) of the hollow sphere from amphiphilic chitosan derivatives can be calculated with the dox concentration . herein , encapsulation efficiency was obtained as described below : wherein , a is the total amount of the dox , and b is the amount of dox remaining in the supernatant . the calculation results are shown in fig2 , wherein x - axis is the substitution degree of hydrophobic group , and y - axis is encapsulation efficiency . as shown in fig2 , as the substitution degree of hydrophobic group and the length of the side chain of the hydrophobic group are increased , the encapsulation efficiency also is improved . the improved encapsulation efficiency is caused by the increase of the hydrophobic interaction of inner shell of the hollow sphere , which can restrict the outward diffusion of dox from the hollow sphere . hence , the release behavior of drugs can be controlled by adjusting the substitution degree and the length of side chain of the hydrophobic group . drug release behavior of the hollow sphere from amphiphilic chitosan derivatives a solution of dox and dox - amphiphilic chitosan derivative complex prepared with the amphiphilic chitosan derivatives in embodiments 1 - 4 was dialyzed against 0 . 1 m buffer ( 100 ml ) under 37 ° c . the dox concentration in the buffer was measured at various times over a 7 - day period to detect the drug release behavior of the hollow spheres from the amphiphilic chitosan derivatives . the results are shown in fig3 , wherein x - axis is the substitution degree of hydrophobic group , and y - axis is the releasing percentage of the dox . as shown in fig3 , dox is not easily released from the hollow spheres while the substitution degree of the hydrophobic group is increased , so that it is possible to release drugs for a long time . hence , the drug release behavior of the hollow spheres can be controlled by adjusting the substitution degree of the hydrophobic group . therefore , the hollow sphere from the amphiphilic chitosan derivatives of the present invention can be a suitable candidate for delivery of anticancer drugs . in conclusion , the amphiphilic chitosan derivatives with hydrophilic and hydrophobic modification of the present invention can form hollow a sphere by self - assembling , wherein the size of the hollow sphere is about 20 - 200 nm , and the hollow sphere has a double layer structure , i . e . hydrophilic - hydrophobic - hydrophilic configuration . hence , comparing with the chitosan hollow sphere prepared by template - coating or layer - by - layer building , the process of hollow sphere from chitosan derivatives is simpler and cheaper . also , the hollow sphere from the amphiphilic chitosan derivatives of the present invention is formed through the self - assembling , so the hollow sphere can capsulate drug without destroying the drug activity . in addition , the hollow sphere from the amphiphilic chitosan derivatives of the present invention not only can maintain the stability of the active component , but also can improve the encapsulation efficiency of the hollow sphere . furthermore , in order to control the encapsulation efficiency and the drug release behavior of the hollow sphere from the amphiphilic chitosan derivatives of the present invention , the critical aggregation concentration and surface character of the hollow sphere can be adjusted through modifying the kinds , substitution degree , and the length of the side chain of the hydrophobic group . therefore , the hollow sphere from the amphiphilic chitosan derivatives of the present invention can be used in substitution for the liposome , and serve as the drug carriers for anticancer chemotherapy . although the present invention has been explained in relation to its preferred embodiment , it is to be understood that many other possible modifications and variations can be made without departing from the scope of the invention as hereinafter claimed .
a hollow sphere from amphiphilic chitosan derivatives and a method of preparing an amphiphilic chitosan derivative complex for medical use are disclosed , and the hollow sphere from amphiphilic chitosan derivatives comprises : chitosan derivatives represented by the following formula , which self - assemble and form a hollow sphere in a solvent ; wherein , each r 1 is independently hydrogen , c 1 ˜ c 4 alkyl , c 1 ˜ c 6 carboxyl , sulfate group , or phosphate group , each r 2 is independently hydrogen , c 1 ˜ c 12 alkyl , c 1 ˜ c 6 carboxyl , or c 2 ˜ c 12 acyl group , and m is an integer of 100 - 2000 .