Enzyme-Triggered Intestine-Specific Targeting Adhesive Platform for Universal Oral Drug Delivery

Patient adherence to chronic therapies can be suboptimal, leading to poor therapeutic outcomes. Dosage forms that enable reduction in dosing frequency stand to improve patient adherence. Variation in gastrointestinal transit time, inter-individual differences in gastrointestinal physiology and differences in physicochemical properties of drugs represent challenges to the development of such systems. To this end, a small intestine-targeted drug delivery system is developed, where prolonged gastrointestinal retention and sustained release are achieved through tissue adhesion of drug pills mediated by an essential intestinal enzyme catalase. Here proof-of-concept pharmacokinetics is demonstrated in the swine model for two drugs, hydrophilic amoxicillin and hydrophobic levodopa. It is anticipated that this system can be applicable for many drugs with a diverse of physicochemical characteristics.

experiments were prepared by protein precipitation and a simple derivatization protocol adapted from the method presented by Junnotula and Licea-Perez [1].Stock solutions of each compound were prepared in methanol at a concentration of 500 μg/mL containing H3PO4 (1%v/v).A twelve-point calibration curve was prepared in black swine serum ranging from 10-25000 ng/mL.100 µL of each sample was spiked with 50 µL of 500 ng/mL levodopa-d3 in acetonitrile as internal standards for levodopa.Then 400 µL of fluorescamine at 5mg/mL in acetonitrile was added to each sample.Samples were centrifuged for 10 minutes at 13,000 rpm.300 µL was pipetted into fresh Eppendorf tubes and allowed to incubate covered at 37 °C for 60 minutes.Following incubation, 200 µL of supernatant was pipetted into a 96-well plate containing 200 µL of water.Analyte concentrations of levodopa and deuterated internal standard were analyzed using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS).Analysis was performed on a Waters ACQUITY UPLC®-I-Class System aligned with a Waters Xevo® TQ-S mass spectrometer (Waters Corporation, Milford MA).Liquid chromatographic separation was performed on either an Acquity UPLC® CSH (50mm × 2.1mm, 1.7 μm particle size) column at 50 °C.Sample introduction and ionization was by electrospray ionization (ESI) in the positive ionization mode.10.0 µL was injected onto the instrument for analysis.For the analysis of levodopa and deuterated internal standards, the mobile phase consisted of aqueous 0.1% formic acid, 10mM ammonium formate solution (Mobile Phase A) and acetonitrile: 10 mM ammonium formate, 0.1% formic acid solution (95:5 v/v) (Mobile Phase B).The mobile phase had a continuous flow rate of 0.45 mL/min for using a time and solvent gradient composition.The initial composition, 95% Mobile Phase A, was held for 1.00 minutes.Following which, the composition was changed linearly to 5% Mobile Phase A and 95% Mobile Phase B until 1.25 minutes.The composition was held constant at 95% Mobile Phase B until 3.00 minutes.At 3.25 minutes the composition returned to 95% Mobile Phase A, where it remained for column equilibration for the duration of the run, ending at 4.00 minutes.Waters MassLynx 4.1 software was used for data acquisition and analysis.The mass to charge transition (m/z) used to quantitate levodopa fluorescamine was 458.16>139.137and 461.16>232.4 for levodopa-d3 fluorescamine.

Swine serum sample preparation
Samples containing amoxicillin in swine serum from in vivo experiments were prepared for LC-MS/MS by protein precipitation.Stock solutions of amoxicillin and internal standard moxifloxacin were prepared in methanol at a concentration of 500 μg/mL.A twelve-point calibration curve was prepared in blank swine serum ranging from 2.5-10000 ng/mL.100 µL of each sample was spiked with 200 µL of 250 ng/mL moxifloxacin in acetonitrile as the internal standard.Samples were centrifuged for 10 minutes at 13,000 rpm.Following centrifugation, 200 µL of supernatant was pipetted into a 96-well plate containing 200 µL of water.Analyte concentrations of amoxicillin were analyzed using Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS).Analysis was performed on a Waters ACQUITY UPLC®-I-Class System aligned with a Waters Xevo® TQ-S mass spectrometer (Waters Corporation, Milford MA).Liquid chromatographic separation was performed on either an Acquity UPLC® BEH (50mm × 2.1mm, 1.7 μm particle size) column at 50 °C.Sample introduction and ionization was by electrospray ionization (ESI) in the positive ionization mode.10.0 µL was injected onto the instrument for analysis.For the analysis of amoxicillin, the mobile phase consisted of aqueous 0.1% formic acid, 10mM ammonium formate solution (Mobile Phase A) and acetonitrile: 10 mM ammonium formate, 0.1% formic acid solution (95:5 v/v) (Mobile Phase B).The mobile phase had a continuous flow rate of 0.6 mL/min using a time and solvent gradient composition.The initial composition, 100% Mobile Phase A, was held for 1.00 minutes.Following which, the composition was changed linearly to 50% Mobile Phase A and 50% Mobile Phase B until 1.25 minutes.At 1.50 minutes the composition changed to 20% Mobile Phase A, and at 2.50 minutes the composition was 100% Mobile Phase B. The composition was held constant at 100% Mobile Phase B until 3.00 minutes.At 3.25 minutes the composition returned to 100% Mobile Phase A, where it remained for column equilibration for the duration of the run, ending at 4.00 minutes.Waters MassLynx 4.1 software was used for data acquisition and analysis.The mass to charge transition (m/z) used to quantitate amoxicillin was 366.264>114.112and 402.21>110.12 for internal standard moxifloxacin.Representative histology images of rat tissues and pig small intestine.For the control group (left), SD rats were orally gavaged with the same volume of water (n = 5) daily for 28 days, and for the Test group (right), SD rats were orally gavaged with ALG-DA pill ingredients (60 mg per kg rat, n = 5) daily for 28 days.Scale bar, 100 μm or 200 p, (A) Representative histology images of rat brain, (B) Representative histology images of rat heart, (C) Representative histology images of rat liver, (D) Representative histology images of rat spleen, (E) Representative histology images of rat lung, (F) Representative histology images of rat kidney, (G) Representative histology images of rat stomach, (H) Representative histology images of rat small intestine, (I) Representative histology images of rat colon, (J) Representative histology images of pig small intestine.Pills were put on the small intestine for 6 hours via laparotomy.Scale bar, 1mm.

Figure S 2 .
Figure S 2. Size test of polymerization of dopamine.(A) Graphs of polymerization of

Figure S 4 .
Figure S 4. Gelation test of oxidation products of alginate-dopamine.(A) The gelation of oxidation reaction products of alginate-dopamine (ALG-PDA) and alginate-dopamine in tris base buffer, H2O2 and catalase at a concentration of 2% (W/V).(B) The reaction of pills of ALG-PDA after 10 min attachment to the small intestine.

Figure S5 .
Figure S5.Adhesion test of oxidation product of alginate-dopamine.Digital photographs of adhesion on the small intestine of alginate-dopamine and oxidation product of alginate-dopamine using water flushing at different time-points.

Figure S7 .
Figure S7.The biocompatible of alginate-dopamine hydrogel to various cell lines.(A) MTT assay of the proliferation of cells cultured with alginate-dopamine hydrogel for 6 hours (n =4).(B) MTT assay of the proliferation of cells cultured with alginate-dopamine hydrogel for 24 hours (n =4).Dotted lines indicate 80% and 90% survival rate.

Figure S8 .
Figure S8.Weight of rats.Rats of control group were orally gavaged with water (n = 5) daily for 1 month, and rats of drug group were orally gavaged with pill formulation ingredients (60 mg per kg rat, n = 5) daily for 28 days.

Figure S9.
Figure S9.Representative histology images of rat tissues and pig small intestine.For the control group (left), SD rats were orally gavaged with the same volume of water (n = 5) daily for 28 days, and for the Test group (right), SD rats were orally gavaged with ALG-DA pill ingredients (60 mg per kg rat, n = 5) daily for 28 days.Scale bar, 100 μm or 200 p, (A) Representative histology images of rat brain, (B) Representative histology images of rat heart, (C) Representative histology images of rat liver, (D) Representative histology images of rat spleen, (E) Representative histology images of rat lung, (F) Representative histology images of rat kidney, (G) Representative histology images of rat stomach, (H) Representative histology images of rat small intestine, (I) Representative histology images of rat colon, (J) Representative histology images of pig small intestine.Pills were put on the small intestine for 6 hours via laparotomy.Scale bar, 1mm.TableS1.Biochemical indexes of rat blood after rats were gavaged with water (n = 5) and pill formulation ingredients daily for 28 days.

Table S 1
. Biochemical indexes of rat blood after rats were gavaged with water (n = 5) and pill formulation ingredients daily for 28 days.Complete blood counts Test.Rats were gavaged with water (Control, n = 5) and pill ingredients (Test, n=5) daily for 28 days.