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Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-Assembled Nanoparticles for Oral Delivery of Insulin Wei Shan, Xi Zhu, Min Liu, Lian Li, Jiaju Zhong, Wei Sun, Zhirong Zhang, and Yuan Huang ACS Nano, Just Accepted Manuscript • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 7, 2015
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Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-Assembled Nanoparticles for Oral Delivery of Insulin
Wei Shan, Xi Zhu, Min Liu, Lian Li, Jiaju Zhong, Wei Sun, Zhirong Zhang and Yuan Huang*
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Block 3,Southern Renmin Road, Chengdu 610041, PR China
E-mail address for corresponding author:
[email protected] 1
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ABSTRACT. Nanoparticles (NPs) have demonstrated great potential for the oral delivery of protein drugs that have very limited oral bioavailability. Orally administered NPs could be absorbed by the epithelial tissue only if they successfully permeated through the mucus that covering the epithelium. However, efficient epithelial absorption and mucus permeation require very different surface properties of a nanocarrier. We herein reported self-assembled NPs for efficient oral delivery of insulin by facilitating both of these two processes. The NPs possessed a nanocomplex core composed of insulin and cell penetrating peptide (CPP), and a dissociable hydrophilic coating of N-(2-hydroxypropyl) methacrylamide copolymer (pHPMA) derivatives. After systematic screening using mucus-secreting epithelial cells, NPs exhibited excellent permeation in mucus due to the “mucus-inert” pHPMA coating, as well as high epithelial absorption mediated by CPP. The investigation of NP behavior showed that the pHPMA molecules graduated dissociated from the NP surface as it permeated through mucus, and the CPP-rich core was revealed in time for subsequent transepithelial transport through the secretory endoplasmic reticulum/Golgi pathway and endocytic recycling pathway. The NPs exhibited 20-fold higher absorption than free insulin on mucus-secreting epithelium cells, and orally administered NPs generated a prominent hypoglycemic response and an increase of the serum insulin concentration in diabetic rats. Our study provided the evidence of using pHPMA as dissociable “mucus-inert” agent to enhance mucus permeation of NPs, and validated a strategy to overcome the multiple absorption barriers using NP platform with dissociable hydrophilic coating and drug-loaded CPP-rich core.
KEYWORDS. Oral delivery, insulin, nanoparticles, mucus, epithelium, cell penetrating peptide
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Protein therapeutics are increasingly being applied for the treatment of various diseases in clinic due to their high therapeutic efficacy and excellent selectivity.1 Despite the frequent dosing that is required for patients with many diseases, especially chronic ones, the administration of these drugs are largely limited to parenteral routes, and thus causes problems including low patient compliance and safety issues.2 The oral bioavailability of these biomolecules is very limited (< 1%) due to their inherent low permeability across the epithelium and the rapid indigestive degradation.3 The advance of nanotechnology and nanomedicine in the past decade has opened a new perspective for oral delivery of biomolecules. Numerous nanocarriers have been reported for the oral delivery application, including such as liposomes, nanogels and polymeric nanoparticles (NPs).4, 5 Improved bioavailability was achieved with these nanocarriers utilizing different absorption strategies, such as paracellular permeation through opened tight junctions and transcytosis mediated by certain receptors.6, 7
Different from the NPs developed for parenteral application, the behavior of orally administered NPs is greatly influenced by their interaction with the mucus that covers and protects the underlying epithelium.8 Intestinal mucus is a layer of slippery secretion in constant and fast renewal, and thus can rapidly trap and remove foreign particles, especially those possessing cationic and hydrophobic nature in surface property.9, 10 Reduced absorption of NPs caused by the mucus barrier has recently been paid with increasing attention and different techniques have been proposed to solve this problem.8,
10-13
One excellent example is the development of Mucus Penetrating
Particles (MPP) inspired by the virus that can freely diffuse through mucus.8 It was found that NPs
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with a densely PEGylated surface exhibited excellent diffusion ability in mucus. The hydrophilic and electrically neutral surface of the NP serves as “mucus-inert” interface that prevents the trapping of the particles, and facilitates their diffusion through the low viscosity pores in mucus matrix.10 However, it should be noted that mucus permeation and epithelial absorption requires very different surface properties of a nanocarrier. As reported by different researchers, the hydrophilic and electro-neutral surface may prevent the interaction of NPs with the target cell membrane and thus may decrease their uptake by epithelial cells.14-16 Therefore, a nanocarrier designed for oral delivery should have the ability to conquer the obstacles of both mucus layer and epithelium.
Cell-penetrating peptides (CPPs) are short peptides that can facilitate cellular internalization of various molecular cargos.17 CPPs were also demonstrated to improve the transepithelial transport of different proteins in the forms of covalent conjugation15 or physical mixture.18 Previous study of our group
demonstrated
that
nanocomplex
(NC)
prepared
with
penetratin
(CRQIKIWFQNRRMKWKK), one of the most promising CPPs, effectively transported encapsulated insulin across epithelium.19 However, the cationic property of CPPs, which plays a vital role in their cellular internalization,20 also induced a high affinity of CPP-rich particles with the negatively charge mucin that formed the mucus matrix, and thus reduced their absorption efficiency.
We herein developed a self-assembled NP for oral delivery of protein drug, which was designed with a novel strategy to achieve both excellent mucus permeation and transepithelial absorption. Using insulin as a model drug, the NPs possessed a CPP/protein nanocomplex core and
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an N-(2-hydroxypropyl) methacrylamide (HPMA) polymer (pHPMA) derivatives coating. HPMA polymer has been explored as a hydrophilic macromolecular carrier for chemotherapeutic agents for decades, 21-24 and at least six HPMA-based therapeutics have currently progressed into phase I or phase II clinical trials.25, 26 In our study, HPMA copolymer was validated for the first time as a dissociable “mucus-inert” coating material, which assembled on the NP surface to facilitate mucus permeation, while separated in time for subsequent CPP-mediated transport through epithelium. With the screening of pHPMAs with different charge density, and the investigation of the NP behavior in mucus and on the mucus-secreting epithelial cell, we demonstrated that the NP with CPP-rich core and dissociable pHPMA coating could successfully solve the dilemma of choosing between high mucus permeation and high epithelial transport.
RESULTS
HPMA polymer synthesis. Different from other commonly used hydrophilic polymers (e.g. PEG), the physiochemical properties of pHPMA can be easily tuned by manipulating the monomers. pHPMA derivatives were synthesized using radical solution polymerization method with negatively charged N-methacryloyl-glycylglycine (MA-GG-OH) monomer. The MA-GG-OH monomer was fed at three different ratios (5%, 10% and 20%) in order to endow the pHPMA with different densities of negative charge, and the polymers were termed as pHPMA-1, pHPMA-2 and pHPMA-3, respectively (Figure 1A). The 1H nuclear magnetic resonance (1H-NMR) spectra and mass spectrograms were recorded (Supplementary Information Figure S1~5). The molecular weight
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of the all pHPMA derivatives were ~ 45 kDa with a PDI less than 1.8 (Supplementary Information Table S1). The charge density of three polymers was measured using titration method and was determined to be 0.28, 0.43, and 1.05 mmol/g, respectively (Figure 1B).
Preparation and characterization of nanoparticles. The nanoparticles are prepared through a two-step approach based on self-assembly strategy. Penetratin is a poly-cationic peptide that possesses strong affinity with negatively charged proteins.19 Aqueous solution of insulin and penetratin were first mixed at a weight ratio of 2:1 to form polyelectrolyte nanocomplexes (NCs). Then, by adding the NCs into a pHPMA solution, the positively charged NC and negatively charged pHPMA were spontaneously assembled to form NPs with nanocomplex core surrounded by pHPMA coating, as shown schematically in Figure 1C. NPs prepared with three pHPMAs were termed accordingly as NPs-1, NPs-2 and NPs-3, and another batch of NPs (NPs-4) were prepared with a higher amount of pHPMAs-3 in formulation (Table 1). The TEM images of NCs and NPs were shown in Figure 1D. The NCs exhibited a size of 148 nm as tested by dynamic light scattering, while the size of all pHPMA coated NPs was approximately 175 nm (Figure 1E). NPs in mucus were reported to transport primarily through lower viscosity pores within the elastic matrix, and the mesh spacing ranges approximately from 10 nm to 200 nm.10 Therefore, the NPs that were sub-200nm in diameter could meet the sterical requirement for rapid diffusion. Besides, all NP exhibited insulin encapsulation efficiency above 80% and drug loading efficiency above 40%. The encapsulated insulin was released in a sustained manner within 10 h in phosphate buffered saline (pH 7.4) (Supplementary Information Figure S6).
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Fig 1. (A) Chemical structure of HPMA polymer derivatives with different ratio of MA-GG-OH monomer. (B) Charge densities of the three pHPMAs as measured by titration method. Data are means ± SD (n= 3). (C) Schematic diagram of NP, which possessed a penetratin/insulin nanocomplex core with sheddable pHPMA coating. (D) TEM images of NCs, NPs-1 and NPs-2. (E) Size and zeta potential of NCs and different NPs. Data are means ± SD (n= 3). (F) Emission spectrum of free F-insulin (a), NPs (b) and T-pHPMA (c) with excitation at 440 nm. Table 1. Characterizations of NCs and NPs. Data are means ± SD (n= 3). Sample
pHPMA
Concentration
Size (nm)
PDI
EE%
DL%
NCs
-
-
148.3±5.1
0.31±0.09
95.3±1.6
65.6±4.0
NPs-1
pHPMA-1
2 mg/mL
173.4±2.7
0.22±0.02
94.4±1.7
48.5±6.7
NPs-2
pHPMA-2
1 mg/mL
177.3±15.2
0.24±0.03
94.9±1.1
52.6±1.5
NPs-3
pHPMA-3
1 mg/mL
174.4±7.2
0.26±0.03
89.1±3.5
49.9±7.6
NPs-4
pHPMA-3
2 mg/mL
176.7±9.8
0.24±0.02
83.8±4.4
44.4±5.6 7
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As shown in Figure 1E, the CPP-rich NCs were highly positively charged with zeta potential of 20.9 mV. After the pHPMA coating, the all NPs possessed a negative zeta potential that ranging from -1.47 mV to -28.2 mV. Lower surface charge was observed with NPs prepared with pHPMA of higher charge density (NPs-2 and NPs-3) or with higher amount of pHPMA in the formulation (NPs-4). The reversal of surface charge suggested the successful coating of anionic pHPMA on the outer surface of the NPs. The coating of pHPMA was further validated using a fluorescence resonance energy transfer (FRET) analysis.27 The NPs were prepared with TRITC labeled HPMA polymers (T-pHPMA) and FITC labeled insulin (F-insulin), which formed a FRET pair. The emission intensity of F-insulin decreased at 520 nm and that of TRITC increased at 573 nm, which implied the energy transfer from donor to acceptor (Figure 1F). The FRET efficiency between two interacting partners was 38.8% and the FRET distance was calculated to be 5.7 nm, which again suggested the formation of pHPMA coated NPs. Moreover, the enzymatic stability of the insulin encapsulated in the particles was investigated using trypsin and α-chymotrypsin. All NPs exhibited better protection for loaded insulin against digestive enzyme compared with NCs or free insulin (Supplementary Information Figure S7, 8).
Mucus permeation ability of NPs. The mucus layer has evolved to protect the body with excellent ability to immobilize and remove cationic and hydrophobic molecules and particles.10, 28 The negative charges of carboxyl or sulfate groups on the mucin proteoglycans and the periodic hydrophobic globular regions along mucin strands allow efficient formation of multiple low-affinity
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adhesive interactions with the cationic and hydrophobic regions on the surfaces of foreign substances.10 Since major types of proteins have both hydrophilic and hydrophobic region in their structures, and CPPs also possess strong poly-cationic character, the CPP-protein complex would exhibit high affinity with the mucin. We hypothesize that by concealing the nanocomplex beneath the negatively charged hydrophilic HPMA polymer, the particle could exhibit reduced interaction with the mucus layer. To test our hypothesis, we first evaluated the interaction of the particles with mucin by measuring the amount of particles-mucin aggregates formed in different concentration of mucin. As shown in Figure 2A, significant lower amounts of aggregates were formed with all tested NPs compared with the insulin-CPP NCs. For example, at mucin concentration of 0.5% (w/v), the aggregates formed in NPs-1 groups were only 15.4% relative to that of NCs. However, no difference was observed among the four tested NPs. As all NPs with HPMA coating possessed hydrophilic surface with negative charge, both of the electrostatic and hydrophobic interaction between NPs and mucin can be avoided. Therefore, higher absolute value of the negative change cannot further reduce the interaction. Then we tested the permeation of the NPs through the mucus layer using an Ussing Chamber System. Porcine intestinal mucus was amounted on a semi-penetrating membrane, which separated the donor and acceptor compartments. The apparent permeability (Papp) values were calculated based on the accumulative amount of diffusion within 3 h (Figure 2B). Interestingly, although all NPs exhibited low affinity with mucin, the permeation ability decreased for NPs with higher absolute value of the surface charges. The NPs-1 exhibited the highest amount of permeation, which was 2-fold as that of NCs, whereas the permeation of NPs-4 was even lower than NCs. This phenomenon might due to the electrostatic repulsion between the 9
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highly negatively charged NPs with the mucus, and correlated with those studies regarding MPP, in which electro-neutral surface was demonstrated as beneficial factor for mucus penetration.
Fig 2. (A) The amount of particle-mucin aggregates formed at different concentration of mucin. The fluorescent intensity of aggregates for NCs at 0.5% mucin presented as control and normalized to 100%. Data are means ± SD (n= 3), ★p
