Influence Of Combination/modification With Borneol On BBB Penetration Of Poly(Amidoamine) Dendrimers In Vitro

ObjectiveBlood-brain barrier(BBB)plays vital role in maintaining the homeostasis and normal function of central nervous system(CNS).But more than 98%of small molecular-weight drugs and nearly 100%of large molecular-weight drugs cannot penetrate the BBB,affecting the therapeutic efficacy of many CNS diseases.According to the basic theories of traditional Chinese medicine,borneol(BOR)is often called an "upper guiding drug",which can guide other components to the targeting tissues or organs in the upper part of the body,especially in the brain.It was reported that the BBB opening triggered by BOR was a physiological process and did not damage the brain tissue.Although BOR has been proved to be capable of promoting drugs into the brain efficiently,there have been few reports about the effect of BOR on the transport of nano-carriers across the BBB.Poly(amidoamine)(PAMAM)dendrrimers are highly branched macromolecules that have specific size,shape and chemical functionality.In present study we chose generation 5 PAMAM(PAMAMG5)dendrimers as a nano-carrier model to explore the influence of combination or modification with BOR on the distribution of nano-carriers to the brain.Methods?PEG3K-PAMAMG5 dendrimers with four grafting ratios were synthesized by the reactions which were carried out at the molar ratios of PEG3K to PAMAMG5 from 16:1 to 64:1.FT-IR and 1H-NMR were used to confirm the structures and modification degrees of PEG3K on the dendrimers.The hydrodynamic diameters and Zeta potential were measured by dynamic light scattering(DLS).In addition,the hemolysis assay and cytotoxicity analysis were performed to evaluate the influence of the different grafting ratios of PEG3K on the biocompatibility of the dendrimers.Cellular uptake was studied using fluorescence-activated cell sorting(FACS).?PEG3K-PAMAMG5 was labeled with fluorescein isothiocyanate(FITC)and the resulting dendrimers was characterized by 1H-NMR.The MTT assay was used to evaluate the cytotoxicity of PEG3K-PAMAMG5 combined with BOR against HBMECs.The uptake of PEG3K-PAMAMG5 alone or combined with BOR by HBMECs was measured by FACS.The influence of combination with BOR on BBB penetration of PEG3K-PAMAMG5 was evaluated on a BBB model in vitro.?DOX was selected as a model drug and was entrapped in PEG3k-PAMAMG5(DOX@PEG3k-PAMAMG5).The hydrodynamic size and Zeta potential of DOX@PEG3k-PAMAMG5 were investigated by DLS.In vitro cytotoxicity and cellular uptake were explored by MTT assay and FACS.A BBB/C6 co-cultured model was developed to study the effect of combination with BOR of DOX@PEG3k-PAMAMG5 on anti-tumor activity after transporting across the BBB.Anti-glioma effect in vivo of DOX@PEG3k-PAMAMG5 combined with BOR was assessed by tumor volume of C6 glioma-bearing mice.?Succinic anhydride was chosen as a linker to modify the primary amino groups on the surface of PAMAMG5 with BOR.The resulting dendrimer(BOR-PAMAMG5)was characterized by FT-IR and 1H-NMR.The pharmaceutics properties and cytotoxicity in vitro of BOR-PAMAMG5 were investigated.In addition,DOX@BOR-PAMAMG5 was prepared and loading drug content and encapsulation efficiency were determined.The MTT assay was used to evaluate the anti-tumor activity of DOX@BOR-PAMAMG5 against C6 cells.A BBB monolayer model in vitro was established to explore BBB penetration of DOX@BOR-PAMAMG5.Results?The results of FT-IR and 1H-NMR analysis showed that four PEG3K-PAMAMG5 dendrimers were successfully synthesized.The conjugated numbers of PEG3K were estimated to be 10,18,26 and 31,when the feed molar ratios of PEG3K and PAMAM G5 were 16:1,32:1,48:1 and 64:1,named as 10PEG3k-PAMAMG5,18PEG3k-PAMAMG5,26PEG3k-PAMAMG5 and 31PEG3k-PAMAMG5 respectively.PEG3k-PAMAMG5 dendrimers exhibited increased particle size and decreased Zeta potential as compared with unmodified PAMAMG5.The hemolytic toxicity of PEG3k-PAMAMG5 dendrimers significantly dropped compared with PAMAMG5(P<0.01)and the hemolysis rate decreased with the increase of the grafting ratio of PEG3k.In contrast,the cytotoxicity of them showed a similar trend.Flow cytometry quantitative results unveiled that there were remarkably lower amounts of the uptake of PEG3k-PAMAMG5 dendrimers by HBMECs compared with that of PAMAMG5(P<0.01)and the amounts obviously decreased with the numbers of PEG3k(P<0.01 for 10PEG3k-PAMAMG5,18PEG3k-PAMAMG5 and 31PEG3k-PAMAMG5;P<0.05 for 26PEG3k-PAMAMG5 and 31PEG3k-PAMAMG5).Therefore,compared with the other PEG3k-PAMAMG5 dendrimers,31PEG3k-PAMAMG5 had lower cytotoxicity and HBEMCs cellular uptake which was more suitable to explore the influence of BOR combination on improving the transport of nano-carriers across the BBB.?PEG3k-PAMAMG5 was successfully labeled with FITC and1H-NMR analysis showed the conjugated number of FITC was 5.2.MTT assay proved that BOR didn't display cytotoxicity at concentration of 1.0-200.0 ?g·mL-1 in HBMECs.The uptake of PEG3k-PAMAMG5 alone or combined with BOR by HBMECs had no obviously difference and the latter even had minor inhibition.On the BBB model in vitro,combination with BOR significantly enhanced the transport ratio of PEG3k-PAMAMG5 compared with alone after 4 h incubation with HBMECs(P<0.05).These results suggested that combination with BOR could boost the transport across the BBB of nano-vectors and loosing the tight junction of BMECs maybe was one of the main mechanisms.?DOX@PEG3k-PAMAMG5 was prepared,the mean particle size and Zeta potential of which were(140.67± 1.64)nm(most of the particles were mainly around 23 nm)and(17.73± 1.00)mV respectively.The encapsulation efficiency and drug loadings were(54.4±1.57)%and(2.50±0.08)%.Co-incubation with BOR had little influence on C6 inhibition and HBMECs uptake of DOX@PEG3k-PAMAMG5 during 4 h.On the HBMECs/C6 co-cultured model in vitro,compared with alone,the combination of BOR(0.1 mg·mL-1)increased the inhibition of C6 cells of DOX@PEG3k-PAMAMG5(DOX 20 ±mol·L-1),which had obvious difference(P<0.05).The C6 glioma-bearing mouse model was used to evaluate the influence of combination of BOR on anti-tumor efficacy in vivo and the results showed that DOX@PEG3k-PAMAMG5 combined with BOR showed a better anti-glioma effect than DOX@PEG3k-PAMAMG5.The above experiments results indicated further that combination with BOR could enhance BBB penetration of drug-loaded nano-carriers which maybe also achieved by looseing the tight junction of BMECs.?FT-IR and 1H-NMR showed that BOR-PAMAMG5 was synthesized successfully and almost 47 BOR were modified on the periphery of each PAMAMG5.The mean particle size and Zeta potential of BOR-PAMAMG5 were(146.3±0.42)nm(most of the particles was mainly around 34.67 nm)and(16.60±0.32)mV respectively.MTT assay demonstrated that BOR-PAMAMG5 had lower cytotoxicity than unmodified PAMAMG5 at concentration of 0.001-100.0 ?mol·mL-1 to HBMECs and C6 cells,which meant that modification with BOR could reduce the cytotoxicity of PAMAMG5 by decreasing the positive potential of PAMAMG5 surface.In addition,DOX@BOR-PAMAMG5 was prepared and the encapsulation efficiency and drug loading were(6.50±0.51)%and(64.25±4.22)%(n=3)respectively.On the BBB model in vitro,BBB penetration of DOX@BOR-PAMAMG5 had time-dependence and compared with DOX@PAMAMG5,DOX@BOR-PAMAMG5 showed more significantly increase across the BBB(P<0.01).These results suggested that modification of PAMAMG5 with BOR could lower the cytotoxicity and significantly promote nano-carriers transport across the BBB.ConclusionOur results showed combination of PEG3k-PAMAMG5 with BOR had no obviously difference in the uptake by HBMECs but enhanced BBB penetration significantly(P<0.05),which demonstrated that combination of BOR could improve the transport of nano-carriers across the BBB and loosing the tight junction of BMECs might be the main mechanisms.On the other hand,our synthesized BOR-PAMAMG5 had lower toxicity and significantly higher BBB transportation(P<0.01)than unmodified PAMAMG5,which suggested modification of PAMAMG5 with BOR could significantly promote nano-carriers transport across the BBB.