protein drugs and poorly water-soluble drugs

European Journal of Pharmaceutics and Biopharmaceutics 82 (2012) 219–229

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European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

Review Article

pH-sensitive polymeric nanoparticles to improve oral bioavailability of peptide/protein drugs and poorly water-soluble drugs Xue-Qing Wang a, Qiang Zhang a,b,⇑ a b

Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing, China The State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China

a r t i c l e

i n f o

Article history: Received 23 March 2012 Accepted in revised form 23 July 2012 Available online 3 August 2012 Keywords: pH-sensitive polymeric nanoparticles Oral bioavailability Absorption improvement Peptide and protein drugs Poorly water-soluble drugs

a b s t r a c t pH-sensitive polymeric nanoparticles are promising for oral drug delivery, especially for peptide/protein drugs and poorly water-soluble medicines. This review describes current status of pH-sensitive polymeric nanoparticles for oral drug delivery and introduces the mechanisms of drug release from them as well as possible reasons for absorption improvement, with emphasis on our contribution to this field. pH-sensitive polymeric nanoparticles are prepared mainly with polyanions, polycations, their mixtures or cross-linked polymers. The mechanisms of drug release are the result of carriers’ dissolution, swelling or both of them at specific pH. The possible reasons for improvement of oral bioavailability include the following: improve drug stability, enhance mucoadhesion, prolong resident time in GI tract, ameliorate intestinal permeability and increase saturation solubility and dissolution rate for poorly water-soluble drugs. As for the advantages of pH-sensitive nanoparticles over conventional nanoparticles, we conclude that (1) most carriers used are enteric-coating materials and their safety has been approved. (2) The rapid dissolution or swelling of carriers at specific pH results in quick drug release and high drug concentration gradient, which is helpful for absorption. (3) At the specific pH carriers dissolve or swell, and the bioadhesion of carriers to mucosa becomes high because nanoparticles turn from solid to gel, which can facilitate drug absorption. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Oral administration is the most desirable way among all the drug delivery routes because of its high patient compliance. If Abbreviations: AUC, area under the concentration–time curve; CHC, carboxymethyl–hexanoyl chitosan; Cmax, maximal blood concentration; CyA, cyclosporine A; CyA-E100 nanoparticles, CyA loaded Eudragit E 100 nanoparticles; CyA-L100 nanoparticles, CyA loaded Eudragit L 100 nanoparticles; CyA-L100-55 nanoparticles, CyA loaded Eudragit L 100-55 nanoparticles; CyA-S100 nanoparticles, CyA loaded Eudragit S 100 nanoparticles; CyA-HP50 nanoparticles, CyA loaded HP-50 nanoparticles; CyA-HP55 nanoparticles, CyA loaded HP-55 nanoparticles; Eudragits, poly(methacrylic acid-co-ethylacrylate) copolymers; Eudragit E100, basic butylated methacrylate copolymer; Eudragit L100, methacrylic acid–methyl methacrylate copolymer (1:1); Eudragit L100-55, methacrylic acid–ethyl acrylate copolymer (1:1) Type A; Eudragit S100, methacrylic acid–methyl methacrylate copolymer (1:2); FITC-insulin, fluorescein isothiocyanate labeled insulin; GLP-1, glucagon-like peptide-1; HPMCP, hydroxypropyl methylcellulose phthalate; HP50, one type of hydroxypropyl methylcellulose phthalate which dissolves at pH 5.0; HP55, one type of hydroxypropyl methylcellulose phthalate which dissolves at pH 5.5; HPMCAS, hydroxypropyl methylcellulose acetate succinate; MAA–EA, methacrylic acid–ethyl acrylate; NP, nanoparticles; PBS, phosphate buffered saline; PCP, polymethacrylic acid–chitosan–polyethylene glycol; PLGA, poly(lactic-co-glycolic) acid; P(MAA-gEG), poly(methacrylic acid-g-ethylene glycol); Rho, rhodamine 6G; TEER, transepithelial electric resistance; TP5, thymopentin (Arg-Lys-Asp-Val-Tyr). ⇑ Corresponding author. Department of Pharmaceutics, School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. Tel./fax: +86 10 82802791. E-mail address: [email protected] (Q. Zhang). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2012.07.014

successful, it will solve the current noncompliance-related problems associated with injections of peptide and protein drugs and also improve the bioavailability of poorly water-soluble medicines. The advantages of oral delivery system have led to many attempts to develop oral formulations for peptides or proteins such as thymopentin [1] and insulin [2–6], and poorly water-soluble medicines such as cyclosporine A [7,8], tacrolimus [9], HIV-1 protease inhibitor CGP 57813 [10] and CGP 70726 [11]. As it is well known, the luminal pH varies from highly acidic in the stomach (pH, 1.2–3.0) to slightly basic in the intestine (pH, 6.5– 8.0). Such pH variations can cause pH-induced oxidation, deamidation or hydrolysis of peptide and protein drugs, leading to loss of their activity [12]. Besides pH, there are abundant enzymes, such as pepsin, trypsin, chymotrypsin, procarboxypeptidase, pancreatic amylase and pancreatic lipase in the GI tract [13]. Most peptides and proteins are sensitive to the enzymes. To overcome the damages from the pH change or enzyme digestion, nanoparticles are widely used for the oral delivery of active agents. Nanoparticles are colloidal carriers ranging in size from 10 to 1000 nm, in general smaller than 200 nm. Their controlled release and protection properties of the compounds of interest make them very advantageous in the scope of drug delivery applications [14]. Nowadays, stimuli-sensitive nanoparticles that respond to pH, temperature and magnetic fields are paid much attention [15–18].

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As a kind of stimuli-response nanoparticles, pH-sensitive nanoparticles develop rapidly. According to their characters, they can be basically divided into two types. One induces drug release at higher pH due to ionizable functional groups on the polymer backbone or side chain, for example, nanoparticles prepared with poly(methacrylic acid) [19], hydroxypropyl methylcellulose phthalate [8] and poly(acrylic acid) grafted poly(vinylidenefluoride) [20]. This type of nanoparticles is usually used for oral drug delivery [1–11], where a physiological pH shift in GI tract facilitates the swelling or dissolution of the carriers. The other type of pH-sensitive nanoparticles has a reversed swelling or dissolution behavior from the former. It swells under acidic conditions and can be used to target tumors, lysosomes and endosomes, where pH is relatively low [21]. Recently, a novel drug delivery system named pH-sensitive nanomatrix was prepared in our laboratory [22,23]. The pH-sensitive nanomatrix was made from the pH-sensitive polymethylacrylate and nano-porous silica already used in pharmaceutical processes. It has successfully improved the absorption of poorly water-soluble drugs and peptides. In this review, we will focus on the pH-sensitive nanoparticles and nanomatrix used for oral delivery system. First, pH-sensitive nanoparticle drug delivery systems prepared from different polymers will be described, followed by the discussion of their drug release characteristic. Second, the mechanisms of drug release from pH-sensitive nanoparticles will be reviewed. Finally, the possible reasons for pH-sensitive nanoparticles to improve the bioavailability of oral administrated drugs will be explored, with emphasis on our contribution to this field.

2. Different pH-sensitive nanoparticle drug delivery systems and their pH-sensitive drug release characteristics 2.1. pH-sensitive nanoparticles prepared from polyanions 2.1.1. pH-sensitive nanoparticles prepared from Eudragits Poly(methacrylic acid-co-ethylacrylate) copolymers (trade name: Eudragits) are commonly used for coating of tablets and preparation of controlled release formulations. Eudragits are divided into several types, such as basic butylated methacrylate copolymer, methacrylic acid–methyl methacrylate copolymer, methacrylic acid–ethyl acrylate copolymer and ammonio methacrylate copolymer. Basic butylated methacrylate copolymer (Eudragit E100) is a cationic copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate, which can dissolve in stomach. Methacrylic acid–methyl methacrylate copolymer (1:1) (Eudragit L100) and methacrylic acid–methyl methacrylate copolymer (1:2) (Eudragit S100) are anionic copolymers based on methacrylic acid and methyl methacrylate. Eudragit L100 dissolves when the pH is higher than 6, while Eudragit S100 dissolves when the pH is higher than 7. Methacrylic acid–ethyl acrylate copolymer (1:1) Type A (Eudragit L100-55) contains an anionic copolymer based on methacrylic acid and ethyl acrylate, which dissolves when the pH is higher than 5.5 [24]. Gurny and colleagues [10,11,25,26] first introduced several poorly water-soluble drugs, CGP 57813, CGP 70726 and RR01, into pH-sensitive nanoparticles. CGP 57813, CGP 70726 and RR01 are all highly lipophilic compounds, almost insoluble in water. Although they possess excellent in vitro activity and acceptable membrane permeation capacity, they have poor oral bioavailability in vivo. When they were incorporated into Eudragit L100-55 or S100 nanoparticles, their bioavailability was improved significantly. Dai and coworkers [7] adopted different types of Eudragits (E100, L100-55, L100 and S100) as pH-sensitive polymers to prepare cyclosporine A (CyA) loaded nanoparticles and investigated the bioavailability and pharmacokinetics of CyA pH-sensitive

nanoparticles, comparing with the currently available microemulsion preconcentrate formulation (Neoral). The mean blood levels of CyA after oral administration of a single dose of each type of nanoparticles and Neoral are shown in Fig. 1. CyA-S100 nanoparticles showed the highest maximal blood concentration (Cmax) and the highest area under the concentration–time curve (AUC), while CyA-E100 nanoparticles showed the lowest Cmax and the lowest AUC. The relative bioavailability of CyA in the groups of CyAE100, CyA-L100-55, CyA-L100 and CyA-S100 nanoparticles were 94.8%, 115.2%, 113.6% and 132.5%, respectively (Table 1). Fig. 2 indicates the characteristic of CyA release from the Eudragit nanoparticles. The release profiles of Eudragit E100, L100-55, L100 and S100 nanoparticles exhibited significant pH sensitivity. The amounts of CyA released from CyA-S100 and CyA-L100-55 nanoparticles in the medium pH 6 5.5 were less than 25.8% and 38.2%, respectively, while these values were more than 83.6% and 96.3% from CyA-L100 and CyA-E100 nanoparticles, respectively. Nowadays, Eudragits are extensively used for pH-sensitive nanoparticles preparation [9,27,28]. To control the drug release precisely, the nanoparticles prepared with Eudragits usually mixed with some other polymers [1,2,29]. For example, Makhlof [29] prepared a budesonide-loaded pH-sensitive nanosphere formulation for colon-specific delivery using polymeric mixtures of poly(lactic-co-glycolic) acid (PLGA) and Eudragit S100. The nanospheres showed strongly pH-dependent drug release properties in acidic and neutral pH values followed by a sustained release phase at pH 7.4. Animal experiments revealed the superior therapeutic efficiency of budesonide-loaded nanospheres in alleviating the conditions of trinitrobenzenesulfonic acid-induced colitis model. The in vivo studies using coumarin-6-loaded nanospheres displayed higher colon levels and lower systemic availability of the fluorescent marker when compared with simple enteric-coating. Moreover, quantitative analysis of the fluorescent marker and confocal laser scanning studies showed strong and specific adhesion of the nanospheres to the ulcerated and inflamed mucosal tissue of the rat colon. 2.1.2. pH-sensitive nanoparticles prepared from HPMCP Hydroxypropyl methylcellulose phthalate (HPMCP), available in market since 1971, has been widely used as an enteric-coating agent by pharmaceutical industry. Two types of HPMCP, HP50 and HP55, which dissolve at pH 5.0 and 5.5, respectively, are usually used [30]. Wang and her colleagues [8] prepared CyA loaded nanoparticles with HPMCP by solvent displacement method. The 3000

Blood Concentration of CyA (ng/ml)

220

2500

Neoral

2000

CyA-E100 CyA-L100-55 CyA-L100 CyA-S100

1500

1000

500

0 0

10

20

30

40

50

60

70

80

Time (h) Fig. 1. Blood concentration profiles of CyA after oral administration of CyA loaded Eudragit E100, L100-55, L100, S100 nanoparticles and the reference Neoral microemulsion into fasted SD rats at a dose of 15 mg/kg. Reproduced from Ref. [7] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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X.-Q. Wang, Q. Zhang / European Journal of Pharmaceutics and Biopharmaceutics 82 (2012) 219–229 Table 1 Examples of bioavailability enhancement of cyclosporine and insulin after oral administration of different pH-sensitive nanoparticles. Active substances

pH-sensitive nanoparticle types

Relative bioavailability of CyA or Insulin

Ref.

Cyclosporine

CyA-E100 nanoparticles CyA-L100-55 nanoparticles CyA-L100 nanoparticles CyA-S100 nanoparticles CyA-HP50 nanoparticles CyA-HP55 nanoparticles CyA-chitosan nanoparticles

94.8% vs. Neoral in rats 115.2% vs. Neoral in rats 113.6% vs. Neoral in rats 132.5% vs. Neoral in rats 82.3% vs. Neoral in rats 119.6% vs. Neoral in rats 173% vs. Neoral in beagle dogs

[7] [7] [7] [7] [8] [8] [33]

Insulin

PLGA-HP55 nanoparticles

6.27% vs subcutaneous injection of insulin solution in diabetic rats 14.9% vs. subcutaneous injection of insulin solution in diabetic ratsa 15.1% vs. subcutaneous injection of insulin solution in diabetic rats 20.1% vs. subcutaneous injection of insulin solution in diabetic rats

[5]

Chitosan nanoparticles Chitosan and poly(g-glutamic acid) nanoparticles Chitosan and poly(g-glutamic acid) nanoparticles filled in enteric-coated capsules a

[34] [48] [41]

Pharmacological bioavailability.

Fig. 2. In vitro drug release profiles from CyA pH-sensitive nanoparticles and the reference Neoral microemulsion by ultracentrifuge method. Reproduced from Ref. [7] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

relative bioavailability of CyA-HP50 and CyA-HP55 nanoparticles in rats were 82.3% and 119.6%, respectively (Table 1), compared with that of Neoral. The bioavailability of CyA-HP55 nanoparticles was significantly higher than that of CyA-HP50 nanoparticles. Same as Eudragits, the release characteristic of CyA-HP50 and CyA-HP55 nanoparticles was also different. Most of CyA was released from HP50 nanoparticles even at pH 2.0. While in HP55 nanoparticles, the release of CyA was little at pH 2.0, there was a sudden increase in CyA release at pH 4.0–5.0 [8]. Cui [5] prepared PLGA nanoparticles and PLGA-HP55 nanoparticles as drug carriers for orally insulin delivery by a modified emulsion solvent diffusion method. The relative bioavailability of PLGA nanoparticles and PLGA-HP55 nanoparticles compared with subcutaneous injection (1 IU/kg) in diabetic rats was 3.68% and 6.27% (Table 1), respectively. The initial release of insulin from the nanoparticles in simulated gastric fluid over 1 h was 50.5% and 20.0%, respectively, showing obviously pH-dependent character. 2.2. pH-sensitive nanoparticles prepared from polycations The cationic polymer used to prepare pH-sensitive nanoparticles is mainly chitosan. It is the second most abundant polymer in nature after cellulose, with a linear structure consisting of 1–4 linked

2-acetamido-2-deoxy-b-D-glucopyranose and 2-amino-b-D-glucopyranose units [31,32]. Chitosan exhibits good biocompatibility, biodegradability, mucoadhesiveness and permeation enhancing effect, and it is widely used to prepare nanoparticles. El-Shabouri [33] prepared chitosan nanoparticles for improving the oral bioavailability of CyA by emulsification solvent diffusion method. The AUC0–24h of CyA in nanoparticle group was 1.8-fold that of Neoral in beagle dogs (Table 1). Insulin-loaded chitosan nanoparticles were prepared by ionotropic gelation of chitosan with tripolyphosphate anions [34]. The insulin association was found up to 80%, and its in vitro release showed a great initial burst with a pH sensitivity. Furthermore, after administration of 21 IU/kg insulin in the chitosan nanoparticles, the hypoglycemia was prolonged over 15 h and the average pharmacological bioavailability relative to subcutaneous injection of insulin solution was up to 14.9% (Table 1). However, as a weak polybase due to the large quantities of amino groups on its chain, chitosan can dissolve easily at low pH, while it is insoluble at higher pH ranges [31]. This property facilitates its use in the delivery of chemical drugs to the stomach. But for the delivery of drugs to the intestine, the solubility property of chitosan encounters some limitations. To make it a suitable matrix for oral drug delivery, different chitosan derivatives with favorable properties are developed, with improved functioning also in a higher pH. These modified chitosan derivatives include carboxylated chitosan [35], N-trimethylated chitosan [36], carboxymethyl–hexanoyl chitosan (CHC) [37], etc. Liu [37] prepared an amphiphilic chitosan derivative, CHC, which had excellent swelling ability and water solubility under natural conditions because of the introduction of carboxymethyl and hexanoyl substitution. In this work, the influences of the degree of carboxymethyl and hexanoyl substitution on the pH-sensitive swelling behavior and drug release behavior from CHC hydrogels (cross-linked with genipin) were studied. It was found that the pH sensitivity was more pronounced in CHC than in N,O-carboxymethyl chitosan because the hexanoyl group altered the state of water in CHC by inhibiting intermolecular hydrogen bonding. In addition, greater pH sensitivity was observed in samples bearing longer hydrophobic chains (carboxymethyl–palmityl chitosan). When ibuprofen (a poorly water-soluble therapeutic agent) was used as a model drug, the burst release of ibuprofen was less prominent in the CHC with high degree of carboxymethyl substitution. 2.3. pH-sensitive nanoparticles prepared from the mixture of polyanions and polycations Some techniques using the advantages of both polyanions and polycations have been developed [1,2,38–42]. Most of the

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(A) 100

Insulin chitosan-Eudragit L100-55 nanoparticles Insulin released (%)

80

60

40

20

0

pH1.0 pH2.5 pH4.0 pH5.8 pH6.4 pH7.4

(B) 100

insulin chitosan-Eudragit L100 nanoparticles Insulin released (%)

80

60

40

20

0

pH1.0 pH2.5 pH4.0 pH5.8 pH6.4 pH7.4

(C)

100

Insulin released (%)

nanoparticle systems related are composed of the positive-charged chitosan and a negative-charged polymer, such as Eudragit [2,38,43], poly(g-glutamic acid) [39–42], alginate [44], methacrylic acid [45] and polyaspartic acid [46]. Since the nanoparticles can be prepared by using two oppositely charged polymers, the need of cross-linker and homogenizer is not considered as an absolute requirement. This provides a mild procedure that is beneficial to prevent protein denaturation [38]. Zhang [1,2,43] developed a chitosan–Eudragit system to incorporate peptides. As a model drug, thymopentin (TP5, ArgLys-Asp-Val-Tyr) and insulin were incorporated. For TP5, two types of nanoparticles were prepared: chitosan nanoparticles and chitosan–Eudragit S100 nanoparticles. In chitosan nanoparticles, TP5 showed a burst release (45% in 60 min) and a 100% of drug release at 24 h when it was incubated in 0.1 N HCl. While the release slowed down at pH 7.4 phosphate buffered saline (PBS) and pH 5.0 PBS, the maximal release percentage was 85.5% or 60.7% at 24 h, respectively. However, the maximal release percentage of TP5 from chitosan–Eudragit S100 nanoparticles was 24.6% in 0.1 N HCl, 41.0% in pH 5.0 PBS and 81.4% in pH 7.0 PBS media at 24 h, respectively [1]. For insulin [2,43], besides chitosan, different Eudragits (L100-55, L100 and S100) were used as negatively charged polymer. The insulin release from three types of nanoparticles showed obvious pH-sensitiveness [2] (Fig. 3). Jelvehgari [38] also reported a chitosan–Eudragit system to incorporate insulin. Nanoparticles were formed by complex coacervation method using Eudragit L100-55 and chitosan with various molecular mass. The results showed that with the increasing molecular mass of chitosan, the zeta potential of nanoparticles and the release rates of insulin were increased. Sung [39–41,47,48] reported another pH-responsive nanoparticle system composed of chitosan and poly(g-glutamic acid). Insulin and aspart-insulin were loaded into this system. Oral administration of insulin-loaded nanoparticles showed a significant hypoglycemic action in diabetic rats, and the corresponding relative bioavailability of insulin was approximately 15.1% (Table 1) [48]. The biodistribution study in a rat model showed that some of the orally administered nanoparticles were retained in the stomach for a long duration, which might lead to the disintegration of nanoparticles and degradation of insulin. To overcome these problems, they developed freeze-dried nanoparticles and filled them in an enteric-coated capsule. Upon oral administration, the entericcoated capsule remained intact in the acidic environment of the stomach, but dissolved rapidly in the proximal segment of the small intestine. The relative bioavailability of insulin was found to be approximately 20.1% (Table 1) [41]. Aspart-insulin, a rapidacting insulin analogue, was also loaded into the pH-responsive nanoparticle system [39]. The biodistribution results obtained in the single-photon emission computed tomography/computed tomography study indicated that the orally administered aspartinsulin was absorbed into the systemic circulation, while the drug carrier (chitosan) was mainly retained in the gastrointestinal tract. Via the subcutaneous route, the peak aspart-insulin concentration in the peripheral tissue/plasma was observed at 20 min after injection. Within 3 h, half of the initial dose of aspart-insulin was degraded and excreted into the urinary bladder. In contrast, via oral delivery, there was constantly circulating aspart-insulin in the peripheral tissue/plasma during the course of the study, while 20% of the initial dose of aspart-insulin was metabolized and excreted into the urinary bladder. In the pharmacodynamic and pharmacokinetic evaluation in a diabetic rat model, the orally administered aspart-insulin-loaded nanoparticles produced a slower hypoglycemic response for a prolonged period of time, whereas the subcutaneous injection of aspart-insulin produced a more pronounced hypoglycemic effect for a relatively shorter duration.

Insulin chitosan-Eudragit S100 nanoparticles

80

60

40

20

0

(D)500

pH1.0 pH2.5 pH4.0 pH5.8

pH6.4 pH7.4

Insulin chitosan-Eudragit L100-55 nanoparticles

400

Particle size (nm)

222

300

200

100

0

pH1.0 pH2.5 pH4.0 pH5.8

pH6.4 pH7.4

Fig. 3. Insulin released from three types of chitosan–Eudragit nanoparticles and mean particle sizes of insulin chitosan–Eudragit L100-55 nanoparticles in different dissolution media at 0.5 h. Adapted from Ref. [2] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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2.4. pH-sensitive nanoparticles prepared from cross-linked polymers (nanogels) Besides the nanoparticles prepared with linear polymers introduced above, cross-linked polymers (nanogels) are of special interest in controlled release applications for the oral delivery system. Nanogels have three-dimensional network structures. Under acidic conditions, the polymers formed collapsed networks as a result of hydrogen bond forming, but ionized and swelled upon raising the pH, thereby allowing the release of the entrapped drug. Nanogels are usually prepared by cross-linking the commonly used polymers, such as polyacrylic acid, polymethacrylic acid, polyethylene glycol and chitosan [6,49,50], with different cross-linkers or in different free radical polymerization reactions. Methacrylic acid–ethyl acrylate (MAA–EA) [50] cross-linked with diallyl phthalate is synthesized via the emulsion polymerization process. The polymers are insoluble lattices at low pH. By increasing the pH, ionization of acid groups enhances, which increases the solubility and electrostatic repulsion between polymeric chains, yielding changes in particle interaction potential. In vitro release studies were performed at varying pH (at the pH of 5, 6, 7.4 and 8) for procaine hydrochloride loaded nanogels (with MAA-EA molar ratio of 50:50 and cross-linked density of 4 wt.%). The percentage of procaine hydrochloride released at the pH of 8 was about 90% compared to 30% at the pH of 5. Poly(methacrylic acid-g-ethylene glycol) (P(MAA-g-EG)) [51,13] is also a pH-sensitive material. Sipahigil [51] prepared P(MAA-gEG) particles by free radical solution polymerization of methacrylic acid and poly(ethylene glycol). Model drugs such as diltiazem HCl, diclofenac Na, ciprofloxacin HCl and isoniazid were embedded in the particles. The particles controlled the release rate of small molecular weight model drugs according to the pH of the medium. The in vitro release profile of the drugs from P(MAA-g-EG) particles in gradually changing pH buffers (pH, 1.2, 5.8, 6.8 and 7.4) showed obvious pH dependence. There was no drug released at pH 1.2. As the pH increased, the complexes dissociated, allowing the network to swell, and rapid drug release occurred. pH-sensitive polymethacrylic acid–chitosan–polyethylene glycol (PCP) nanoparticles were prepared under mild aqueous conditions via polyelectrolyte complexation and free radical polymerization reaction [6]. Insulin and bovine serum albumin as model peptides and proteins were incorporated into the nanoparticles by diffusion filling method. PCP nanoparticles exhibited good protein encapsulation efficiency and pH-responsive release profile in vitro. Alginate is a water-soluble linear polysaccharide extracted from brown sea weed. It is composed of alternating blocks of 1–4 linked a-L-guluronic and b-D-mannuronic acid residues. In theory, alginate shrinks at low pH (gastric environment), and the encapsulated drug may not release [44]. But there are some limitations for alginate to deliver drug into the GI tract because alginate dissolves rapidly at higher pH, which will cause burst release of entrapped protein drug once it enters the intestine. To overcome these limitations, George [52] prepared cross-linked alginate-guar gum matrix. The release profiles of bovine serum albumin from test hydrogels were studied under simulated gastric and intestinal media. Protein release from test hydrogels was minimal at pH 1.2 (20%) and significantly higher (90%) at pH 7.4. The presence of guar gum and glutaraldehyde cross-linking increased the entrapment efficiency and prevented the rapid dissolution of alginate in higher pH of the intestine, ensuring a controlled release of the entrapped drug. 2.5. pH-sensitive nanomatrix prepared from Eudragit and nanoporous silica To overcome the problems in stability and scaling up for the nanoparticle colloid system, a novel nanomatrix system for oral

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administration was developed by our group [22,23]. The system was composed of the pH-sensitive Eudragit and nano-porous silica already used in pharmaceutical processes. It was prepared in a relative simple process, rotary evaporation. In the study for fenofibrate [22], two nano-structured SiO2 powder, Aerosil 200 and Sylysia 350, and three pH-sensitive Eudragits were used. Dissolution study showed that different specification of Eudragits and mesoporous silica had a clear effect on the in vitro and in vivo behavior of nanomatrix formulation. Nanomatrix formulations showed faster drug release than crude fenofibrate power and commercial product with micronized fenofibrate. As for different mesoporous silica, nanomatrix prepared with Sylysia 350 released faster than that with Aerosil 200. In the formulations prepared with Sylysia 350 and different Eudragits, Eudragit L100-55 showed the fastest drug release. The optimized nanomatrix system produced a higher drug absorption (AUC0–36h: 1214.3 vs 1070.5, h lg/ml) and a longer mean residence time (10.8 vs 8.2 h) when compared with fenofibrate self-microemulsifying drug delivery system. Fenofibrate existed in a molecular or amorphous state in the nanomatrix, and this state was maintained for up to 1 year, without obvious changes in drug release. The oral bioavailability of optimized nanomatrix after 1 year of storage was similar to that of the freshly prepared form. Some other poorly soluble drugs such as sorafenib [23], CyA and megestrol acetate were also incorporated into the system. Recently, peptides such as insulin [53] and glucagon-like peptide-1 (GLP-1) [54] were tried with the same system. In the insulin formulation, it presented protective function against enzymolysis in vitro, as well as significant hypoglycemic effect in intraperitoneal glucose tolerance tests [53]. As for GLP-1, GLP-1 nanomatrix displayed a 5-fold intestine mucosa permeability and significantly high proteolytic stability compared to native GLP-1. When GLP-1 nanomatrix was orally administrated to rats, its relative bioavailability was 35.7% and hypoglycemic effect reached 77% in comparison with intraperitoneal injection of GLP-1 [54]. Wang [23] compared the relative bioavailability of sorafenib nanomatrix to sorafenib Eudragit S100 nanoparticles; the absorption of sorafenib in nanomatrix was not so high as that in Eudragit S100 nanoparticles (from 16.8% to 40.8%). The reasons may be the decreased dispersion state and bioadhesion activity, or the change in release characteristic because of the adsorbed or the embedded phenomena. The relative bioavailability of sorafenib nanomatrix was 13–33 times to that of sorafenib suspension. Therefore, we think the system is valuable for further study because it has the potential to solve the problems of the nanoparticles, stability and scaling up.

3. The mechanisms of drug release from the pH-sensitive nanoparticles 3.1. Drug burst releases when the nanoparticle carriers dissolve at specific pH conditions The drug release from conventional nanoparticles was mainly by diffusion (Fig. 4A). As for pH-sensitive nanoparticles, they usually exhibited burst release profiles because of the dissolution characters of the carriers. At low pH, the nanoparticles prepared from polycarboxylic acid were solid matrix encapsulating drug, little drug released. As they entered the small intestine, the pH changes from acidic to neutral (6–7.4), carboxylic acid groups deprotonated, the liner polymers dissolved and drugs released quickly (Fig. 4B). To prepare nanoparticles with polyanions and polycations, a proper pH range was needed. Outside of the pH range, the nanoparticles might collapse and release drugs. For example in the chitosan/poly(g-glutamic acid) nanoparticle system

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[40], in the range of pH 2.5–6.6, chitosan and poly(g-glutamic acid) were ionized. The ionized chitosan and poly(g-glutamic acid) could form polyelectrolyte complexes, which resulted in the formation of nanoparticles; However, outside of this pH range (e.g., pH 1.2 and 7.4), the nanoparticles became unstable and subsequently disintegrated. This was because the COO groups on poly(g-glutamic acid) became protonated at lower pH values, while the amine groups on chitosan tended to deprotonate at pH values above 6.5. As for nanomatrix, the drug release process might be more complex than that in the Eudragit nanoparticles. For the system of Sylysia 350 and Eudragit, it was considered as two steps: first, Eudragit dissolved, a small part of drug released; second, with the dissolution of Eudragit, silica exposed and drug embedded in the nano-pores diffused out of the pores and released (Fig. 4C) [23].

favored the release of the drug due to a reduction in diffusion resistance (Fig. 4D). Tan and Tam [55] studied the particle size change with pH during the release process. The size of the particles varied depending on the pH of the dissolution medium and was found to swell at the pH of 7.4 and 8 and de-swell at the pH of 5 and 6. In genipin-cross-linked N,O-carboxymethyl chitosan/alginate hydrogel [44], the swelling ratio was limited (2.5 times) at pH 1.2 due to formation of hydrogen bonds between N,O-carboxymethyl chitosan and alginate. At pH 7.4, the carboxylic acid groups on the genipin-cross-linked N,O-carboxymethyl chitosan/alginate hydrogel became progressively ionized. In this case, the hydrogel swelled more significantly (6.5 times) due to a large swelling force created by the electrostatic repulsion between the ionized acid groups.

3.2. Drug releases when the polymers swell at specific pH conditions

3.3. Drug releases as a result of both polymer dissolution and swelling

Another reason for drug release from nanoparticles was the swelling of the materials [50]. At low pH, the polymers, especially cross-linked polymers, possessed a compact conformation, which significantly reduced the porosity of the matrix. This resulted in a lower release of drug due to the larger resistance for diffusion of the drug out of the nanogel. However, at high pH, the nanogel particles were in a swollen state with a higher porosity that

There was no obvious boundary between dissolution and swelling for the carriers. Some nanoparticle systems might release drug through both the mechanisms (Fig. 4E). Li [2] studied the release of insulin from chitosan–Eudragit L100-55 nanoparticles in vitro (Fig. 3A) and the mean particle size during the release (Fig. 3D). The mean particle size remained unchanged in pH 1.0, 2.5 HCl and 4.0 PBS. When the pH value was elevated to 5.8, the mean

Fig. 4. Drug release mechanisms from conventional and pH-sensitive nanoparticles. (A) Release from conventional nanoparticles by diffusion; (B) release from pH-sensitive nanoparticles after the materials dissolution at specific pH; (C) release from pH-sensitive nanomatrix by two steps, Eudragit dissolves first, a small part of drug released; then silica exposed and drug embedded in the nano-pores diffused out of the pores; (D) release from pH-sensitive nanoparticles after the materials swelling at specific pH; and (E) release from pH-sensitive nanoparticles as the co-effect of dissolution and swelling of the materials at specific pH. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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particle size was increased significantly. These results suggested that at low pH, the nanoparticles were coated by Eudragit L10055, little water permeated into the particles, so the particle size remained unchanged. When the pH value was elevated to 5.8, Eudragit L100-55 dissolved and water infiltrated to the core of the particles. The particle size increased as chitosan swelling and insulin released because of the higher porosity of chitosan.

4. The mechanisms of pH-sensitive nanoparticles for the improvement of oral bioavailability 4.1. Increase saturation solubility and dissolution rate for poorly water-soluble drugs Poor aqueous solubility represents a major hurdle in achieving adequate oral bioavailability for a large percentage of drug compounds in drug development nowadays. The poorly water-soluble drugs pulverized into nanosized particles mean the reduction in the drug particle size down to the submicron range, with the final particle size typically being 100–200 nm or even smaller. The reduction in particle size leads to a significant increase in the solubility and dissolution rate of the drugs, which in turn can lead to substantial increases in bioavailability [56]. Müller and Peters [57] reported that when RMKP 22 (an antibacterial compound) powder was milled from a mean diameter of 2.4 lm to 300 nm, the saturation solubility was approximately doubled. Besides the particle size, the dispersion state of the drug embedded or adsorbed in the particles also influences the solubility. Dai [7] investigated the physical state of both the polymer and drug in the nanoparticle matrices through X-ray powder diffraction studies. The result showed that the original crystal structure of the drug was not found in the pH-sensitive nanoparticles, despite the relatively high drug loading of CyA in the nanoparticles (20%). The absence of crystallinity in the nanoparticles indicated that the drug was amorphous or molecularly dispersed within the polymeric matrices of the nanoparticles, which might enhance the solubility of the poorly water-soluble drugs. Furthermore, Eudragits are also supersaturation stabilizing polymers. They may inhibit nucleation or crystal growth by adsorption on the crystal interface, thereby blocking crystal growth, and supersaturated solution will be obtained [58– 60]. The concentration-enhancing phenomenon can significantly improve drug bioavailability [58,59,61,62].

Fig. 6. CyA amounts in luminal contents of different parts of gastrointestinal tracts (A: stomach; B: duodenum; C: jejunum; D: ileum and E: colon) versus time after oral administration of CyA-S100 nanoparticles (j) and Neoral microemulsion (h) to rats. Reproduced from Ref 64 with permission. Fig. 5. Degradation of TP5 from Eudragit–chitosan nanoparticles in the presence of aminopeptidase N. Reproduced from Ref. [1] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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4.2. Improve drug stability in gastrointestinal tract As previously introduced, the pH in the stomach is 1–3. Some medicines, especially peptides and proteins, are not stable at such low pH condition in stomach. When the drugs were incorporated as nanoparticles, they are protected by the materials from the degradation at acidic pH conditions. Another reason for nanoparticles to improve the stability of the drugs is that the materials protect the drugs from the enzyme degradation in GI tract. Wang and Dai [63] reported the degradation rate of CyA in different gastrointestinal luminal contents and subcellular fractions of different parts of intestine. Among the three formulations (CyA suspension, CyA-S100 nanoparticles and Neoral), CyA suspension degraded most quickly in all tested sections of the GI tract and Neoral group ranked second, while CyA-S100 nanoparticles group was the last, suggesting the significant protective effect of nanoparticles. On the other hand, there was a decreasing sequence of degradation rate of CyA for all formulations in the luminal contents from the top down (stomach ? duodenum ? jejunum ? ileum ? colon). The first reason for this finding might be the change of pH values from stomach to colon. CyA is unstable at extremely low pH like that in stomach, where pH is about 1–3, while in colon, where pH is about 7–8, CyA is rather stable. Another reason for this fact might be the changes in enzyme activity in the GI tract. Pepsin has a high concentration in stomach; however, the enzyme activity

decreased in the lower sections of GI. Among the subcellular fractions of various segments of intestine, there were no obvious differences in the degradation of CyA for the same mucosal subfraction (brush border membrane fraction, cytosol or lysosome) at different segments of intestine (duodenum, jejunum, ileum and colon), possibly due to the similar structure and the function of the mucosal subfraction at different segments of GI. This study indicated that preventing the drug from degradation by the acidic pH and enzymes was important. Zheng [1] reported the results of the enzymatic degradation kinetics of TP5 in the aminopeptidase solution (Fig. 5). Most of TP5 was degraded during one minute, and the half-life of degradation was about 1.5 min when free drug was incubated. In contrast, once TP5 was incorporated into chitosan–Eudragit nanoparticles, the degradation of TP5 by the aminopeptidase was clearly inhibited and the half-life of degradation was markedly prolonged to 15 min. These results indicated that chitosan–Eudragit nanoparticles could effectively protect the incorporated peptide drug from enzymatic degradation in the gut when orally administered. Some nanoparticles not only have the function of protection from enzyme degradation but also have the enzyme inhibitory function. Trypsin inhibitory effect of PCP nanoparticles [6] was studied using casein substrate, and these particles displayed enzyme inhibitory function. For the PCP nanoparticles with a final concentration of 1% (w/v), the inhibition rate was about 50%.

Fig. 7. Fluorescence images of distinct intestinal segments of rats retrieved at 4 h after orally treated with fluorescence-labeled nanoparticles. XY plane: parallel to the intestinal surface; XZ plane: perpendicular to the intestinal surface. Reproduced from Ref 48 with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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4.3. Influence drug transport and distribution in GI tract When drugs are incorporated into nanoparticles, the transport and distribution characters in GI tract may be changed. Wang and Dai [63] investigated the time course of CyA in luminal contents of different parts of GI after CyA-S100 nanoparticles or Neoral was oral administrated to rats. Fig. 6 shows the amount of CyA in luminal contents of different parts of GI tracts. The AUC0.5–24h of CyA-S100 nanoparticles was less than Neoral in stomach but more than Neoral in duodenum, jejunum and ileum. As for the empting rate, the gastric emptying rate of CyA-S100 nanoparticles was 1.88-fold that of Neoral in stomach. From duodenum to colon, the emptying rates of CyA-S100 nanoparticles were all lower than those of Neoral. The fast stomach emptying offered the advantage for CyA-S100 nanoparticles to move into duodenum and jejunum more quickly than Neoral, indicating the lower degradation of CyA in stomach, where pH is 1–3 and the enzyme activity is also high. As demonstrated in the in situ recirculating intestine perfusion experiments, ileum was the main absorption site for CyA-S100 nanoparticles, so the quick movement of CyA-S100 nanoparticles from stomach facilitated the absorption of CyA. Li [43] compared the emptying rate of fluorescein isothiocyanate labeled insulin (FITC-insulin) in GI tract after the oral administration of 2 ml aqueous solution or nanoparticles containing 0.5 mg FITCinsulin. The intestinal emptying rate was 0.89 h 1 for FITC-insulin solution, while it was 0.56 h 1 for FITC-insulin nanoparticles. The slower empting rate in nanoparticle group in intestine prolonged the contacting time of drug to mucosa, which was favorable to the FITC-insulin absorption. 4.4. Improve mucoadhesive characteristics of drugs to mucosa in gastrointestinal tract Wang and Dai [63] reported the mucoadhesive characteristics of CyA nanoparticles. The percent of CyA adsorbed on the mucosa of duodenum, jejunum, ileum and colon indicated that among all parts of the intestine, CyA-S100 nanoparticles exhibited larger mucoadhesive characteristics than Neoral microemulsion, more than two times. This might be the main reason that CyA-S100 nanoparticles increased the absorption of CyA. Since the intestinal mucosa is a lipid membrane, the hydrophobic carrier Eudragit S100 had high affinity to the lipid membrane, which would enhance CyA contacting with gastrointestinal membrane. Wu [64] studied the influence of different carriers on the mucoadhesive characteristics, using Rhodamine 6G (Rho) as model drug. In the system of Eudragit series nanoparticles, the AUC of Rho adhered to different segments in different parts of GI tract was indicated in such a sequence: Rho-Eudragit S100-NP < Rho-Eudragit L100-NP < Rho-Eudragit L100-55-NP. This might result from the different dissolution pH. In every segment, the amount of dissolved Rho had a sequence as: Rho-Eudragit S100-NP < Rho-Eudragit L100-NP < Rho-Eudragit L100-55-NP. So the opportunity of Rho adhered to the mucosa was as the order. In the system of different series polymers, the author compared three materials with same dissolution pH (pH, 5.5), Eudragit L100-55, HP55 and HPMCAS [64]. The AUC of Rho adhered to the stomach was sequenced as: Rho-Eudragit L100-55-NP < Rho-HP55-NP < Rho-HPMCAS-NP. The result indicated that besides the dissolution pH of the materials, other properties related to chemical structure such as electric charge and hydrophobicity could also influence the drug behavior in vivo and the characters of the drug also should not be ignored. Mucoadhesion study on the pH-responsive nanoparticle system composed of chitosan and poly(g-glutamic acid) was conducted on male Wistar rats and examined under a confocal laser scanning microscopy [48]. Fig. 7 shows the fluorescence images of distinct

Fig. 8. Effects of chitosan/poly(g-glutamic acid) nanoparticles on (A) the TEER values of Caco-2 cell monolayers and (B) the cumulative amounts of aspart-insulin transported through Caco-2 cell monolayers at pH 6.6, 7.0 or 7.4. Reproduced from Ref. [39] with permission. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

intestinal segments of rats retrieved at 4 h after orally treated with fluorescence-labeled nanoparticles. The intensity of fluorescence signals observed at the luminal surface of duodenum was stronger and appeared at a deeper level than those seen at the jejunum and the ileum. These results indicated that the nanoparticles could effectively adhere to the mucosal surface and their constituent components were able to infiltrate into the mucosal cell membrane. 4.5. Improve the intestinal permeability As a cationic polysaccharide, chitosan can adhere to epithelial surfaces and open the tight junctions between contiguous cells. It is suggested that the interactions between the positively charged chitosan and the negatively charged portion of the tight junction protein ZO-1 leads to translocation of ZO-1 to the cytoskeleton and subsequently increases the paracellular permeability [65]. Besides ZO-1, Zheng [66] also demonstrated that chitosan nanoparticles could induce strong alterations in the distribution of membrane proteins, fluidity of membrane lipids and general membrane structure. Via the paracellular pathway, Sonaje [39]

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evaluated the chitosan/poly(g-glutamic acid) nanoparticles in opening intercellular tight junctions on Caco-2 cell monolayers. As shown in Fig. 8, test nanoparticles were able to effectively reduce the transepithelial electric resistance (TEER) of Caco-2 cell monolayers (Fig. 8A) and increase the transport of aspart-insulin (Fig. 8B). With increasing pH, chitosan became less positively charged and therefore had a less effect on the intercellular permeability. The observation was pH-dependent, and the amount of aspart-insulin transported was significantly decreased with an increase in pH. It was revealed that the interaction of the positively charged chitosan with the negatively charged sites on cell surfaces and tight junctions induced a redistribution of the tight junction protein ZO-1 and F-actins, thus accompanying the increase in paracellular permeability. 5. Summary Taking advantage of the altered pH gradients in GI tract, pHsensitive nanoparticles have been designed to deliver poorly water-soluble drugs or peptide/protein drugs to different fragments of intestine. pH-sensitive nanoparticles can improve the drug stability, enhance mucoadhesion, prolong resident time in GI tract, ameliorate intestinal permeability and increase saturation solubility and dissolution rate for poorly absorption drugs. As for the advantages of pH-sensitive nanoparticles over conventional nanoparticles, we can conclude as follows: (1) Most carriers have been used as enteric-coating materials for a long time, and their safety has been approved. (2) The carriers dissolve rapidly at specific pH and specific sites, which result in quick drug release and high drug concentration gradient. The phenomenon is helpful for the drug absorption. (3) At the dissolution pH, because the nanoparticles turn from solid state to hydrogel state, the bioadhesion of the carrier to mucosa becomes high at specific fragment, which can facilitate the absorption comparing to conventional nanoparticles. (4) The pH-sensitive nanoparticles can improve the drug stability more effectively. Here, it is worthwhile to mention that the balance among the concentration gradient, absorption opportunity and drug stability is very important. For example, for peptides such as CyA and TP5, stability is an important obstacle, and the main absorption site is the lower part of small intestine. Eudragit S100 can improve its stability effectively, even though the absorption opportunity at the lower part of intestine is limited. On the other hand, for those drugs stable in stomach and upper small intestine, such as CGP 57813 [10], Rhodamine [64] and fenofibrate [22], the absorption opportunity is a key factor, and suitable materials dissolved at lower pH should be selected. In conclusion, pH-sensitive nanoparticles provide great opportunity for water-insoluble or peptide/protein drugs to improve their oral absorption, although there still is long way to go before this potential drug delivery system can finally be used in clinic. Acknowledgment

[3]

[4]

[5] [6]

[7]

[8]

[9]

[10]

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[13]

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[20]

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We thank National Basic Research Program of China (No. 2009CB930300) and State Key Projects (No. 2009ZX09310-001) for their financial support.

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