Effect of Low Molecular Weight Chitosans on Drug Permeation through Mouse Skin: 1. Transdermal Delivery of Baicalin

Effect of Low Molecular Weight Chitosans on Drug Permeation through Mouse Skin: 1. Transdermal Delivery of Baicalin XUEQIN ZHOU,1 DONGZHI LIU,1 HAIYAN LIU,1 QIAOLI YANG,2 KANGDE YAO,2 XUEYAN WANG,3 LEI WANG,3 XINJIAN YANG3 1

School of Chemical Engineering, Tianjin University, Tianjin, China

2

Research Institute of Polymeric Materials, Tianjin University, Tianjin, China

3

Tianjin Changzheng Hospital, Tianjin, China

Received 8 July 2009; revised 17 November 2009; accepted 17 November 2009 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.22063 ABSTRACT: The aim of this work was to evaluate the low molecular weight chitosans (LMWCs) as enhancers of transdermal administration of baicalin, an useful drug for the treatment of atopic dermatitis, viral hepatitis, and HIV infection. Permeation experiments were performed in vitro through mouse skin by using Franz cells. Improved baicalin skin penetration was obtained with the addition of LMWCs or D-glucosamine (b-D-GlcNH2) to the donor solutions. Chitosan molecular weight, degree of deacetylation, pH of donor baicalin solutions, and enhancer concentration all affected LMWC enhancement effects. Significant enhancement was observed at pH 7.0 or 7.5 for CS80-1000, and the enhancement factor (EF) in the codelivery method was calculated as 11.7 or 15.9, respectively. Simultaneously, b-D-GlcNH2 showed greatest enhancement at pH 7.0 with an EF of 11. Moreover, there was an optimal concentration range (0.5–1% by weight for CS80-1000 and 1.0–1.5% for b-D-GlcNH2) to enhance baicalin transdermal delivery. It was concluded that the effective fractions for the enhancement of LMWCs were b-D-GlcNH2 oligomers, and the repeated number of b-D-GlcNH2 was suggested to be in the range 2–6. Enhancement mechanism of LMWCs was also discussed and suggested to be relative to the interactions of LMWC with both baicalin and the lipid of stratum corneum. ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2991–2998, 2010

Keywords: transdermal drug delivery; permeation enhancers; low molecular weight chitosan; baicalin; permeability

INTRODUCTION The transdermal route is an attractive alternative to deliver therapeutic drugs that are subject to extensive first-pass metabolism into the systemic circulation.1 However, the remarkable barrier properties of the skin membrane, especially the outermost stratum corneum layer, cause a poor skin permeability of compounds that are hydrophilic, very lipophilic, of high molecular weight, or charged.2,3 Several strategies have been explored, such as the use of chemical enhancers and of physical techniques. The use of chemical penetration enhancers would be a convenient method. But the problem with most known dermal penetration enhancers is that they are often toxic, irritating, or allergenic.4 Additional Supporting Information may be found in the online version of this article. Correspondence to: Xueqin Zhou (Telephone: 86-22-27400911; Fax: 86-22-27892283; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 2991–2998 (2010) ß 2010 Wiley-Liss, Inc. and the American Pharmacists Association

Chitosan is attracting increasing attention in drug delivery systems due to its excellent biocompatibility, biodegradability, and nontoxicity.5,6 High molecular weight chitosans (HMWCs, unmodified form or chemically/physically modified form) have been found to be good carriers for enhancing skin penetration of effective substances, such as buprenorphine7 and propranolol hydrochloride.8 However, the high molecular weight and low solubility in water or organic vehicles prohibit it from interacting with the underneath layer from the skin surface and thus significantly limit its enhancement effect.9 Fortunately, these drawbacks can be partially circumvented by using low molecular weight chitosan (LMWC), which is water soluble in a wide pH range. Moreover, LMWCs showed superior biological activities compared to HMWCs, including biodegradability, biocompability, antioxidant, and antibacterial activity.10–13 But to date there are few reports on the drug skin permeation effects of LMWCs. Flavonoid glucuronide structure represents a kind of significant effective ingredients in Chineseherbal

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purchased in officinal grade from Xiangxi Chemical Industry (Hubei Province, P.R. China). All other chemicals were of reagent or HPLC grade. Figure 1. Structure scheme of baicalin.

medicine.14,15 Baicalin (Fig. 1) is a flavonoid glucuronide that is isolated from the Chinese traditional medicinal plant Scutellaria baicalensis Georgi (Labiatae). It has antioxidant, antiinflammatory, anti-HIV, and antiangiogenesis activities, induces apoptosis, inhibits formation of colon aberrant crypts, inhibits cancer cell proliferation, protects bone marrow, promotes hemopoiesis, and protects the intestines from adverse drug effects.16–18 Baicalin has been used clinically against atopic dermatitis, viral Hepatitis, and HIV infection, by oral or intravenous formulations.19,20 However, following intravenous administration of baicalin has a short biological half life of 0.16 h21,22 for its relative high molecular weight (MW ¼ 447) and typical physical property (dissociation constant pKa of 2.9, water solubility of 0.091 g/L at 208C and partition coefficient P1-octanol–water of 1.29).23 Furthermore, significant hepatic first-pass metabolism has been observed, and the bioavailability is only 54% after oral administration.24 These undesirable side effects could be offset by using the transdermal route.25,26 However, the skin barrier needs to be overcome. In this article, we investigated the transdermal delivery of baicalin in aqueous solution. LMWCs with different molecular weights were applied to enhance the baicalin transdermal delivery. The effects of pH and LMWC concentration on this enhancement were studied. Finally the enhancement mechanism of LMWCs was discussed in detail.

MATERIALS AND METHODS

Preparation of LMWCS with Different Degree of Deacetylation CS80-1000 (1.016 g) was dissolved in 100 mL of 60% (v/v) methanol aqueous solution. Acetyl anhydride (6, 12, or 18 mL) was added to the mixture and allowed to react at room temperature for 16 h. Then the solution was poured into 200 mL ethanol. The precipitate was separated, washed with ethanol and ethyl ether, and dried in vacuo. The degree of deacetylation of products was measured as reported by Gupta and Jabrail.27 Preparation of Pretreated LMWC Solutions LMWC was dissolved in acetic acid solution (pH 3.0) and the concentration was controlled as 0.5% (by weight). pH value was adjusted exactly with triethanolamine to 6.0, 6.5, 7.0, 7.5, or 8.0. The controls were prepared using the same procedure, except for 0.5% (w/w) acetic acid solution (pH 3.0) that did not contain LMWC. Preparation of Baicalin Solutions Baicalin was dissolved in a solution of glycerol, triethanolamine and deionized water at a ratio of 5:3:25 (v/v) and kept in the dark before use. Preparation of Baicalin-LMWC Solutions Baicalin was dissolved in a solution of glycerol, triethanolamine, and deionized water at a ratio of 5:3:25 (v/v). This solution was added to 0.5% (by weight) acetic acid solution (pH 3.0), with or without LMWC, until the final baicalin concentration reached 100 mg mL1. Finally the pH value was adjusted exactly with triethanolamine or acetic acid to 6.0, 6.5, 7.0, 7.5, or 8.0. The controls were prepared using the same procedure, except for 0.5% (w/w) acetic acid solution (pH 3.0), which did not contain LMWC.

Materials

In Vitro Transdermal Delivery Experiments

LMWCs with molecular weights of 1 kDa (CS801000), 2 kDa (CS80-2000), 4 kDa (CS80-4000), or 5 kDa (CS80-5000) were supplied by Southern Yangtze University (Wuxi, China). The degree of deacetylation was about 80%.27 N-acetylglucosamine (b-D-GlcNAc, >99%) and D-glucosamine hydrochloride (b-D-GlcNH2HCl, >98%) were obtained from Acros Organics (New Jersey, USA). Baicalin contrast product (No. 110715–200212, >99%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Baicalin (97% for injection) was obtained from SiChuan ShiFang RuiBang Natural-Chemical Co. Ltd (SiChuan Province, China). Glycerol was

Kunming mice weighing 25–30 g (supplied and approved by Tianjin Municipal Institute for Drug control) were killed by cervical dislocation and the hair of the abdominal region was carefully shaved. Then a portion (about 1.5  1.5 cm2) of the full thickness of the skin was carefully excised, washed with saline and used afresh. The integrity of the skin was checked upon mounting on the diffusion cells with the aid of a small flashlight. All procedures were approved by Ethics Committee of Tianjin Changzheng Hospital. In vitro skin penetration studies were performed using Franz vertical diffusion cells. The receptor medium consisted of PEG 400 and 0.9% saline at a

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ratio of 40:60 (V/V). Fresh full-thickness, hairless skin (diffusion area, 2 cm2) was mounted in the diffusion cell with the stratum corneum facing the donor cell, and the receptor cell was filled with 16.5 mL receptor medium, which was stirred at 400 rpm and maintained at 37  0.18C. The receptor cells were required to equilibrate at 37  0.18C for 1 h. Air bubbles were then removed. For the co-delivery method: the baicalin-LMWC solutions (2 mL) were placed in the donor compartments and sealed to prevent evaporation. In the pretreatment method: (1) the LMWC solutions (0.5 mL) were added to the donor compartments and kept for 1 h; (2) the LMWC solutions were removed; and (3) the baicalin solutions (2 mL) were placed in the donor compartments and sealed to prevent evaporation. The diffusion process was kept in dark for 24 h. The receptor samples (0.5 mL) were taken at intervals, and the sample volume was simultaneously replaced with fresh receptor medium. The collected receptor samples were analyzed by HPLC.

baicalin solution was added at a known quantity (5 mg mL1). Data Analysis of Permeation Experiments The baicalin accumulative amounts (Qt, mg cm2) at time t are expressed as means  SD (n  3). Q24-0 and Q24-c represent the 24-h baicalin accumulative amounts with or without enhancer, respectively. The permeation fluxes ( F, mg cm2 h1) were deduced from the steady-state slopes of plots of the accumulative amount as a function of time.28 F-0 and F-c represent the baicalin percutaneous permeation fluxes in the presence or absence of enhancer, respectively. To compare the permeation enhancement capacities of penetration enhancers, statistical differences between two mean values (Q24-0 and Q24-c) were evaluated using the unpaired Student’s t-test. If p > 0.05, the results were considered as no significant difference and the enhancement factor (EF) was supposed as 1. If p < 0.05, results were taken as significantly different and the EF was calculated as follows:29

HPLC Analytical Method A Beckman HPLC coupled with system gold software was used to run the entire system. A Beckman C18 ODS column (4.6 mm  250 mm; 5 mm) was used. The mobile phase was 47:53 (v/v) methanol: 0.37% (v/v) phosphoric acid solution, and delivered at a flow rate of 1.0 mL min1. The volume injected was 20 mL. A series of drug solutions, with varying quantities of baicalin ranging from 0.1 to 100.0 mg mL1, were prepared and injected into the HPLC column. The eluent was monitored at 280 nm (retention time, 10.1 min). The peak areas were obtained and subjected to regression analysis. There was a perfect linear relationship (Y ¼ 0.3955 þ 0.98547x) between the concentration of baicalin and its peak area, as indicated by high correlation coefficient (R ¼ 0.99986, p < 0.0001). The inter- and intra-day variation was <2.48%, which indicated high precision of the method. The HPLC method was found to be highly accurate, as shown by a mean recovery of 97.6% when

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EF ¼

F-c F-0

RESULTS Effect of Chitosans with Different Molecular Weight and Deacetylation Degree Chitosan is a copolymer of b-D-GlcNAc and b-DGlcNH2. The bioactive properties of chitosan are dependent on its molecular weight and the degree of deacetylation.30,31 Here, the effects of LMWC molecular weight, together with b-D-GlcNAc and b-DGlcNH2, on the skin penetration of baicalin were investigated (Tab. 1). The baicalin delivery property showed no significant change on the Q24 while using b-D-GlcNAc as an enhancer. In contrast, when using b-D-GlcNH2 as an enhancer, Q24 was increased distinctly, and EF was

Table 1. In Vitro Baicalin Percutaneous Permeation Parameters Obtained from Co-Delivery with LMWCs with Different Molecular Weight or Monomers as Enhancers Enhancer Control b-D-GlcNAc b-D-GlcNH2 CS80-1000 CS80-2000 CS80-4000 CS80-5000

Mean Polymeric Degree of LMWC

Q24a (mg cm2)

F (mg cm2 h1)

EF

— — — 6 12 24 30

9.32  1.24 10.89  0.71 49.32  3.14 100.46  6.92 34.70  2.34 14.46  0.43 9.98  0.81

0.28 0.29 2.21 4.44 1.29 0.32 0.29

— 1 7.9 15.9 4.6 1.1 1

a The data were obtained from transdermal experiments using baicalin aqueous solution with pH of 7.5 and enhancer concentration of 0.5%. The data represent mean  SD (n  3).

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evaluated as 7.9. When CS80-1000 was used, Q24 increased and EF was elevated to 15.9. As the chitosan molecular weight increased up to CS805000, its enhancement effect declined. HMWCs (MW ¼ 80, 150, 300, 400 kDa) were also tried under the same conditions. However, only polyelectrolyte complex precipitation was obtained. Thus, their enhancements were difficult to compare with those of LMWCs. Since the enhancement effects of b-D-GlcNAc and bD-GlcNH2 are so different, one could speculate whether b-D-GlcNH2 or its oligomers are the effective unit. Hence, we decreased the degree of deacetylation of CS80-1000 by reaction with acetyl anhydride, and studied the influence of the degree of deacetylation on the enhancement effect. Figure 2 clearly shows that the enhancement effect of LMWC decreased with reduced degree of deacetylation. When the degree of deacetylation was lowered to 14%, no enhancement effect was observed. The above results indicated that the effective fractions for the enhancement of LWMCs were b-DGlcNH2 oligomers. Since the highest enhancement effect was found with CS80-1000 (molecular weight of 1 kDa and deacetylation degree of 80%), we suggest the repeated number of the effective b-D-GlcNH2 oligomers to be in the range 2–6.

effects of LMWC. The results of baicalin and CS801000 co-delivery under different pH conditions are shown in Figure 3. Most chitosans with pharmaceutical applications, with molecular weights >10 KDa, were indiscerptible in neutral and basic environments. Thus, it is hard to compare their effects in the environments with different pH value.34 Herein, we used water-soluble chitosans with a molecular weight of 1 KDa, and clear chitosan solutions were achieved when pH was <9.0. It was observed clearly that the amount of baicalin accumulation increased steadily from onset, and no lag time was found under different pH conditions, either with b-D-GlcNH2 or CS80-1000 as an enhancer. All curves presented a linear change after 4 h, which indicated that a steady flux was achieved. Table 2 lists the baicalin percutaneous permeation parameters when co-delivered with CS80-1000 under

Effect of pH on Enhancement of CS80-1000 and b-D-GlcNH2 The effective units in LMWCs were b-D-GlcNH2 oligomers, in which the dissociation equilibrium of amino groups is affected strongly by pH.32,33 Hence, pH should be important for the enhancement

Figure 2. Dependence of Q24 (&) and F (*) on deacetylation degree of LMWC with molecular weight of 1 KDa in baicalin co-delivery method. LMWC concentration at 0.5% (by weight). pH value at 7.5. Each Q24 point represents mean  SD (n  3). The horizontal dotted line showed the Q24 of the control. Hence if the Q24 data are on or below this line, they have no significant difference from the control. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

Figure 3. Plots of the baicalin accumulative amounts against the time at different pH conditions with b-D-GlcNH2 (a) and CS80-1000 (b) as enhancer in co-delivery method. The enhancer concentration was controlled at 0.5% by wt. Each point represents mean  SD (n  3). DOI 10.1002/jps

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Table 2. In Vitro Baicalin Percutaneous Permeation Parameters Obtained from CoDelivery with b-D-GlcNH2 or CS80-1000 Under Different pH Conditions Enhancer (Co-Delivery) No enhancer

CS80-1000

b-D-GlcNH2

pH Value

Q24a (mg cm2)

F (mg cm2 h1)

EF

6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0 6.0 6.5 7.0 7.5 8.0

4.89  0.87 3.35  0.59 6.95  0.56 9.33  1.24 11.17  0.84 5.69  0.72 3.71  0.64 76.01  6.48 100.46  6.92 12.18  1.27 20.10  0.07 36.07  9.75 71.14  1.27 49.32  3.14 30.31  0.53

0.14 0.06 0.12 0.28 0.42 0.17 0.07 3.28 4.44 0.44 0.53 1.23 3.02 2.01 1.09

— — — — — 1 1 11.7 15.9 1 2 4.5 11.0 7.6 3.9

a

The enhancer concentration was controlled at 0.5% by weight. The data represent mean  SD (n  3).

different pH conditions. To simplify the study, b-D-GlcNH2 was also investigated in the same environment. When pH was 6.0, b-D-GlcNH2 showed a weak enhancement effect on the baicalin transdermal delivery and EF was evaluated at 2. As pH increased, the enhancement improved, and greatest effect was found at pH 7.0 and EF 11.0. Then, the effect reduced as pH continued to increase. However, different results were obtained in the experiments with CS80-1000 (Tab. 2). When pH was 6.0 and 6.5, there was no significant difference between the Q24 from controls or using CS80-1000 as an enhancer, by the unpaired Student’s t-test. Hence, no significant enhancement was found with pH in this range. Similar results were obtained when pH was 8.0. However, when pH was adjusted to 7.0 or 7.5, distinct enhancements were observed and EF was calculated as 11.7 and 15.9, respectively.

Concentration Effects of CS80-1000 and b-D-GlcNH2 The skin penetration of baicalin was seriously affected by the b-D-GlcNH2 or chitosan concentration. The results of enhancer–concentration–dependent baicalin transdermal delivery are demonstrated in Figure 5. With the increase of b-D-GlcNH2 concentration, the enhancement effect improved first, then decreased when the maximum was achieved. The Q24 data obtained with 2% (by weight) b-D-GlcNH2 as an enhancer did not differ significantly from those in the controls, by the unpaired Student’s t-test. Figure 5 shows that the best enhancement effect was reached when b-D-GlcNH2 concentration was in the range 1.0– 1.5% with the co-delivery method.

Effect of pH on Enhancement of CS80-1000 in Pretreatment Method Figure 4 gives the results obtained in the pretreatment method with CS80-1000 solution in different pH conditions as enhancer. The enhancement results are similar to that obtained from the co-delivery method except that the fluxes were lower than those with the co-delivery method. The maximal EF was calculated as 4.2 at pH 7.5. Herein the CS80-1000 and baicalin solutions were separated, so the enhancement results in pretreatment method, in comparison of the controls, indicated that CS80-1000 could interact with the SC. This result is corresponding to the report by Smith and Wood that chitosans could destroy the skin integrality. And it might be one reason for CS80-1000 to increase the baicalin skin permeability. DOI 10.1002/jps

Figure 4. In vitro Baicalin transdermal delivery at pH 7.5 in the presence of CS80-1000. The data represent mean  SD (n  3). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

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Figure 5. Dependence of baicalin 24 h accumulative amounts with the enhancer concentration. pH value was set at 7.5. The data represent mean  SD (n  3).

The pattern obtained for CS80-1000 is similar: delivery increases to a maximum value and then decreases when enhancer further increases (Fig. 5). The maximum baicalin permeation fluxes were 4.24 and 1.18 mg cm2 h1 at a CS80-1000 concentration of 0.5% for the co-delivery and pretreatment method, respectively. The discrepancy in the optimal enhancer concentration of b-D-GlcNH2 and CS80-1000 (respectively, 1% and 0.5%) for the co-delivery method may be attributed to different effective fractions: b-DGlcNH2 itself for b-D-GlcNH2 while b-D-GlcNH2 oligomers for CS80-1000. Further the difference between the enhancement results for CS80-1000 using the co-delivery and pretreatment methods might have been caused by the interaction of CS801000 with baicalin.

DISCUSSION In the present study, effects of LMWCs on the transdermal delivery of baicalin were investigated. b-D-GlcNAc showed no enhancement effect when codelivered with baicalin, but b-D-GlcNH2 exhibited a clear enhancement with an EF of 7.9. The EF value of LMWCs varies with molecular weight and degree of deacetylation, while the maximum was shown to be 15.9 when using CS80-1000 as an enhancer to codeliver with the baicalin. These results indicated that the effective fractions for the enhancement of LWMCs were b-D-GlcNH2 oligomers. The pH condition of LMWC solution is a key factor relative to its enhancement. As we know, the skin has an isoelectric point (PI) of 4–4.5. Therefore, the carboxylic acid groups of the SC (intracorneocyte keratin and intercorneocyte glycoprotein) are ionized JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 7, JULY 2010

above this pH condition. In the pretreatment method, when the pH is at pH 6.5, most amino groups in CS801000 are protonated for the pKa value of protonated CS80-1000 was about 6.835–37 and should interact with the carboxylic acid groups of the SC strongly as showed in mucosal drug delivery.38,39 But the experimental results under this condition revealed that CS80-1000 has almost no effect on the transdermal delivery of baicalin. On the other hand, when pH is at 7.5, neutral and protonated amino groups coexist for CS80-1000, and the interaction of CS80-1000 with the carboxylic acid groups of the SC was too weak to be detected by 13C-cross polarization/ magic-angle spinning-nuclear magnetic resonance (13C-CP/MAS-NMR) spectroscopy (See Supplements: NMR spectra are similar and there is no significant difference, by the unpaired Student’s t-test, between calculated spin–lattice relaxation times 13CT1 of keratin carbons of the mouse SC keratin before and after treated with CS80-1000 solutions at pH 7.5). However, clear enhancement effect was found for baicalin at pH of 7.5. As we know, SC is composed mainly of proteins (keratin and glycoprotein) and lipids. Thus, we supposed that, the interaction between the CS80-1000 and the SC is very significant for its enhancement effect, and the enhancement mechanism might be concerned primarily with the interaction between the CS80-1000 and the lipid of the SC. Neutral amino groups in CS80-100 would benefit its enhancement effect. Moreover, in the co-delivery method, one could expect that protonated chitosan (cations) might interact via electrostatic coupling with the baicalin anions in the baicalin-LMWC solutions (pH above 6.0) to form a neutral nanocomplex. Several literatures have reported the nanocomplex and electrostatic interaction between protonated chitosan and drug anions, for instance, Ibuprofen and hyaluronic acid. This was confirmed by the determination with X-ray photoelectron spectroscopy (XPS, See Supplements). This interaction should be relatively weak at pH 7.5, but it is enough to intensify the percutaneous permeation property of baicalin, leading to a better baicalin percutaneous permeation property in codelivery method when comparing to that obtained in the pretreatment method. However, the interaction between protonated CS80-1000 and baicalin anion is too weak at pH 8.0 to influence the baicalin transdermal delivery property. Thus, no enhancement effect was observed at pH 8.0. Furethermore, results of b-D-GlcNH2 could further explain the mechanism of LMWCs. The pKa value of b-D-GlcNH2 was reported to be 7.8.36,37 When baicalin is co-delivered with b-D-GlcNH2, most amino groups in b-D-GlcNH2 are protonated at pH 6.0 and hence b-D-GlcNH2 exists primarily in neutral nanocomplex form with baicalin anions. The relative lower DOI 10.1002/jps

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EF value at pH 6.0 (Tab. 2) indicated that neutral nanocomplex could only provide a low enhancement result. When pH was increased to 7.0, protonated b-DGlcNH2 and neutral b-D-GlcNH2 coexist, and neutral b-D-GlcNH2 could interact with the lipid of the SC, inducing an enhanced delivery of b-D-GlcNH2-baicalin nanocomplex. So a relative high EF value of 11.0 was obtained at pH 7.0. When pH was further increased to 7.5 or 8.0, the amount of b-D-GlcNH2baicalin nanocomplex decreased in the donor solutions. The EF value decreased too. This suggested that only interaction between b-D-GlcNH2 and the lipid of the SC is not enough for the enhancement mechanism of LMWCs. Besides, as with azone and oleic acid,40 the efficacy of LMWC appears strongly concentration dependent. There is an optimal concentration range (0.5–1% by weight for CS80-1000 and 1.0–1.5% for b-D-GlcNH2) to enhance baicalin transdermal delivery. The possible explanation is that both CS80-1000 and bD-GlcNH2 would permeate into and remain (then biodegrade or metabolize by the enzymes) within the lipid by interaction with the lipid of SC and lower the lipid barrier for baicalin transport. However, the lipid could hold only a certain amount of enhancers.40 When the enhancer concentration was above the optimal range, extra enhancers, either CS80-1000 or b-D-GlcNH2, would act as a competitor with baicalin or nanocomplex for interacting with the enhancers inside the lipid, resulting in a decreased baicalin transdermal flux. In a summary, LMWCs could enhance baicalin transdermal delivery property. The molecular weight, degree of deacetylation, and pH condition, together with the LMWC concentration, are key factors to influence its enhancement effect. The maximum EF of 15.9 was obtained with CS80-1000 at pH 7.5 with a concentration of 0.5% by weight. Enhancement mechanism of LMWCs should be relative to the interaction between the LMWC and SC, especially the lipid, as well as the nanocomplex between LMWC and baicalin.

ACKNOWLEDGMENTS The authors would like to thank Prof. Xia Wenshui for providing LMWCs, and National Nature Sciences Foundation of China for the financial support (No. 20606024).

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DOI 10.1002/jps