Patent Application
20220296683
Nanoparticle constructs for oral protein delivery must protect and present the payload at the small intestinal epithelium. In a reproducible, scalable, surfactant-free process, a nano-sized core was formed with a model peptide (insulin), in ratios with zinc chloride and L-arginine, which was then cross-linked with silica to form an outer shell. The nanoparticles (SiNP) entrapped insulin in high concentration, preserved its structure, and released it at pH values present in the small intestine. The SiNP delivered insulin to the circulation and reduced plasma glucose in a rat jejunal instillation model. The delivery mechanism required residual L-arginine to act as a permeation enhancer for SiNP-released insulin in the jejunum. The process could be varied in terms of ratios of the core components for entrapping other payloads including the glucagon-like Peptide 1 analogue, exenatide, and bovine serum albumen. The process is the basis of a platform for oral peptide and protein delivery.
Introduction
The number of approved biopharmaceuticals has steadily increased, most of which are injectables1. Oral delivery is the preferred route for biopharmaceuticals due to convenience compared to injections2. Further rationale arises for the estimated 5% of patients who suffer from needle phobia3, whereas others undergo painful injections for specific conditions using low gauge needles4. For insulin and glucagon-like Peptide 1 (GLP-1) analogue treatments for Type II diabetes patients, the rationale to develop oral formats to promote earlier adoption has been advocated in order to achieve better outcomes for patients who are typically reluctant to endure injections before they have to5. Furthermore, the oral route for insulin would mimic the physiological route of delivery to the liver6, would protect β cells of the pancreas, and can potentially reduce side-effects from sub-cutaneous (s.c.) injections, from which only a relatively small proportion of insulin reaches the liver7. Nonetheless, since insulin is a low therapeutic index molecule, low and variable oral bioavailability seen in clinical trials for oral insulin formulations would likely carry risk for patients8. In the current study, insulin was therefore selected as a model peptide. Clinical development of an oral Glucagon-1-like Peptide 1 (GLP-1) analogue is however more advanced, with a daily oral version of semaglutide recently having completed Phase III trials9.
High molecular weight, hydrophilicity, lability, and low intestinal permeability mitigate against delivering peptides orally10. The formulations in clinical trials therefore mostly comprise intestinal permeation enhancers (PEs) and peptidase inhibitors11, 12. Nonetheless, oral peptide bioavailability for such technologies is only ˜1-2% relative to s.c. administration13, 14. Even if efficacy is obtained, there will likely be safety questions relating to intestinal damage, microbiome changes, and association with coeliac and inflammatory bowel diseases in respect of chronic dosing of high doses of enhancers15. As an alternative to use of high doses of enhancers, nanotechnologies can both protect peptides during gastrointestinal (GI) tract transit and, if suitably coated with hydrophilic materials, can enable mucus permeation16, 17. There is considerable debate over whether peptide-loaded nanoparticles are actually taken up by intestinal epithelia to an extent that would account for peptide levels in plasma in vivo. Publications from the EU H2020 consortium, TRANS-INT, tested numerous prototypes, leading to an overall conclusion that in vivo epithelial nanoparticle uptake was low, with most appearing to be either stuck in intestinal mucus, with some associating with the epithelium, e.g.18. Several of these constructs19-21, and indeed two of the original pioneering nanoparticle oral peptide compositions22, 23, comprised excipients known to act as permeation enhancers. A reasonable interpretation therefore is that excipients used as components of the majority of nanoparticle constructs in the literature (with the exception of nanoparticles made with receptor-targeting ligands24, 25), promote uptake of particle-released peptide in the small intestine.
Our hypothesis therefore was that an oral peptide nanoparticle should comprise a simple synthetic scalable process with potential for high loading. In addition, the particle should release its payload in high concentration close to the epithelium, triggered by the higher pH of the small intestine. Intestinal epithelial permeability should be assisted by co-formulated excipients. The components we selected were substances with a history of use in humans in order to alleviate toxicological risk. Silica is attractive as it is biocompatible, FDA-approved, inert, can be functionalized, and has a history of use in large nanoparticle formats in biomedical, food, and food packaging applications26. Moreover, it has already been used in oral peptide nanoparticle constructs either as a core, over which excipients and peptides are attached27, as a core with peptide followed by coating with mucoadhesive28-, muco-permeating-29, and enteric coating polymers30, and also as a mesoporous structure to contain embedded peptides31-33. It can respond to pH changes, so that it is intact in the stomach but releases in the small intestine34. Here, we present a structure in which payloads including human insulin and the GLP-1 analogue, exenatide35, were self-assembled as a core in the presence of the excipients, L-arginine (L-arg), and zinc chloride (ZnCl2). While L-arg was included primarily in order to aid insulin stabilisation36, we noted that L-arg (but not ZnCl2) is an established intestinal permeation enhancer for insulin37. The core particle was then crosslinked with silica to form silica-coated nanoparticles (SiNPs). The resulting SiNPs fulfilled target physicochemical and bioactivity criteria, while batches could be synthesised reproducibly with predictable characteristics. Importantly, the mechanism of achieving insulin delivery across the intestine was based on L-Arg released from the particle enabling permeation of the released insulin in the jejunum.
According to a first aspect of the invention, there is provided a method of preparing a silica-protein sub-micron particle, the method comprising contacting, in an aqueous medium having a pH in the range of about 7.5 to 10 and having an ionic strength in the range of about 1.2 to 60 mM,
Optionally, the protein sub-micron particle cores comprising the basic compound of formula (I) are formed by
Optionally, the protein sub-micron particle cores comprising the basic compound of formula (I) are formed by
Optionally, the basic compound of formula (I) is L or D arginine, or an ester and/or amide thereof.
Optionally, the basic compound of formula (I) is a poly arginine (L or D), or an ester and/or amide thereof.
Optionally, the poly arginine is a dimer or a trimer.
Optionally, the orthosilicic acid or the ester thereof is orthosilicic acid.
Optionally, the orthosilicic acid or the ester thereof is an orthosilicate ester.
Further optionally, the silicate ester retains 0 to 3 hydroxyl groups of the 4 hydroxyl groups of orthosilicic acid; or each hydroxyl group of orthosilicic acid is independently esterified with C1-5 alkyl; optionally, ethyl.
Optionally, each hydroxyl group is esterified with ethyl.
Optionally, the orthosilicic acid or the ester thereof, is present in an amount equivalent to about 10 to 50% (w/w) orthosilicic acid per mg of protein sub-micron particles; wherein, optionally, when the protein is insulin, about 10 to 20% (w/w) orthosilicic acid per mg of protein sub-micron particles is present; and, when the protein is exenatide, about 10 to 50% (w/w) orthosilicic acid per mg of protein sub-micron particles is present.
Optionally, in step (a), about 0.85 to 4.00 mg/mL of the protein is dispersed; wherein, optionally, when the protein is insulin, about 0.85 to 1.5 mg/mL of the protein is dispersed; and, when the protein is exenatide, about 1.0 to 4.00 mg/mL of the protein is dispersed.
Optionally, in step (c), about 0.85 to 4.00 mg/mL of the protein is dispersed; wherein, optionally, when the protein is insulin, about 0.85 to 1.5 mg/mL of the protein is dispersed; and, when the protein is exenatide, about 1.0 to 4.0 mg/mL of the protein is dispersed.
Optionally, the pH of the aqueous medium is in the range of about 8.9 to 9.7.
Optionally, the ionic strength of the aqueous medium is in the range of about 2 to 35 mM.
Optionally, the contacting step is carried out a temperature in the range of about 18 to 40° C.
Optionally, the method further comprises the step of separating the silica-protein sub-micron particle from the aqueous medium by centrifugation.
Optionally, the silica-protein sub-micron particle has a diameter of between about 30 and 600 nm; optionally between about 50 and 300 nm; further optionally between about 80 and 300 nm.
Optionally, the protein is insulin and the silica-protein sub-micron particle has a diameter of between about 80 and 300 nm; or the protein is exenatide and the silica-protein sub-micron particle has a diameter of between about 30 and 600 nm.
According to a further aspect of the invention, there is provided a method of making a GLP-1 receptor agonist sub-micron particle core, the method comprising the steps of:
a. contacting a peptide in an aqueous medium at a pH of about 5.0-8.0 and comprising about 0.3-1.3 mM peptide, wherein the peptide is a GLP-1 receptor agonist,
with
a basic compound of formula (I) or an ester and/or amide thereof:
wherein n is an integer selected from 1 to 10, and p is an integer selected from 1 to 3;
in a molar ratio between the peptide and the basic compound of from 1:about 20 to 1:about 90 to form a step a. reaction mixture having a pH of about 7.5 to 10.0; optionally about 9.5;
b. contacting the step a. reaction mixture with zinc to form a step b. reaction mixture, wherein the ratio of peptide:basic compound:zinc in the step b. reaction mixture is 1:about 20 to 110:9 to 30, optionally between 1:48:16 to 22 and 1:90:20 to 25;
whereby the GLP-1 receptor agonist sub-micron particle core is formed.
Optionally, the GLP-1 receptor agonist is selected from exendin-4, exenatide (Byetta, Bydureon), liraglutide (Victoza), lixisenatide (Lyxumia), dulaglutide (Trulicity) albiglutide (Tanzeum) sitagliptin (Januvia, Janumet, Janumet XR, Juvisync), saxagliptin (Onglyza, Kombiglyze XR), alogliptin (Nesina, Kazano, Oseni), semaglutide (Ozempic) and linagliptin (Tradjenta, Jentadueto); optionally is exenatide or analogues thereof.
Further optionally, the basic compound is arginine and the peptide is exenatide; and the concentration, in the step b. reaction mixture, of Zn is between about 6.5 and about 8.5 mM when the arginine is between about 25 and about 55 mM.
Optionally, the amount of GLP-1 receptor agonist, in the protein sub-micron particle core, is between about 20% w/w and about 80% w/w; optionally between about 30% w/w and about 70% w/w; further optionally between about 35% w/w and about 60% w/w of the protein sub-micron particle core.
According to a further aspect of the invention, there is provided a method of making an insulin sub-micron particle core, the method comprising the steps of:
a. contacting insulin in an aqueous medium having a pH of less than about 6.0 and comprising about 0.50 to about 1.50 mg/mL insulin,
with
a basic compound of formula (I) or an ester and/or amide thereof:
wherein n is an integer selected from 1 to 10, and p is an integer selected from 1 to 3; in a molar ratio between the insulin and the basic compound of from about 1:40 to about 1:140 to form a step a. reaction mixture having a pH of about 7.5 to 10, optionally about 8.0 to 10.0;
b. contacting the step a. reaction mixture with zinc to form a step b. reaction mixture, wherein the molar ratio of insulin:zinc in the step b. reaction mixture is 1:about 0.5 to about 10;
wherein, optionally, the molar ratio of insulin:basic compound:zinc in the step b. reaction mixture is 1:about 66 to about 100:about 3 to about 4.5;
whereby the insulin sub-micron particle core is formed.
Optionally, the insulin in the aqueous medium comprises about 0.9-about 1.2 mg/mL insulin, optionally about 1 mg/mL insulin.
Optionally, wherein n is 1 and p is 3 whereby the basic compound is arginine; and
the concentration, in the step b. reaction mixture, of Zn is about 0.35-0.65 mM, optionally about 0.5 mM, when the arginine concentration is 2 mg/mL; or
the concentration, in the step b. reaction mixture, of Zn is about 0.6-0.9 mM, optionally about 0.75 mM, when the arginine concentration is about 3 mg/mL.
Optionally, the amount of insulin, in the protein sub-micron particle core, is between about 35% w/w and about 95% w/w; optionally between about 45% w/w and about 90% w/w; further optionally between about 50% w/w and about 75% w/w of the protein sub-micron particle core.
Optionally, the insulin is a human insulin or an analog thereof; optionally selected from Lispro (Eli Lilly), Aspart (Novo Nordisk), Glulisine (Sanofi-Aventis), Detemir insulin (Novo Nordisk), Degludec insulin (Novo Nordisk), Glargine insulin (Sanofi-Aventis).
Optionally, the aqueous medium has a pH of less than about 4.0, optionally about pH 2.5.
Optionally, the basic compound is L-arginine.
Optionally, the core comprises the peptide, the basic compound and the zinc; or the core consists of the peptide, the basic compound and the zinc.
Optionally, the method further comprises contacting, in an aqueous medium having a pH in the range of about 7.5 to 10 and having an ionic strength in the range of about 1.2 to 60 mM,
Alternatively, the aforementioned protein sub-micron particle cores are enterically coated in any suitable manner.
As used herein, the term “enteric” refers to a coating that permits transition of the protein sub-micron particle cores through the stomach to the small intestine before the protein is released therefrom. Examples of enteric coating polymers include hydroxypropylmethylcellulose phthalate (HPMCP), hydroxypropylmethylcellulose acetate succinate (HPMCAS) and RL100, and HP-55, which can be coated on the protein sub-micron particle cores and dissolved in the upper intestinal conditions. Other examples include pH-sensitive copolymers of poly(methacrylic acid-co-N-vinyl caprolactam) for the pH-sensitive oral delivery of active ingredients such as insulin and pH-sensitive biodegradable copolymers such as EUDR AGIT® (Evonik) acr ylic drug delivery polymers as an enteric coating polymer that releasesthe protein (such as insulin) only at neutral pH, thereby preventing its degradation at acidic pH.
Optionally, the protein sub-micron particle cores comprising the basic compound of formula (I) are formed by
Optionally, the protein sub-micron particle cores comprising the basic compound of formula (I) are formed by
(c) dispersing 0.5 to 5.5 mg/mL of the protein and the basic compound of formula (I) in an aqueous medium having a pH of about 7.5 to 10 and having an ionic strength in the range of about 1.2 to 60 mM to form the protein sub-micron particle comprising the basic compound of formula (I) in an amount equivalent to 0.2 to 2 mmoles of arginine per mg of sub-micron particles.
The invention also provides a silica-protein sub-micron particle formed by the aforementioned methods, for use in the treatment of diabetes. Optionally, the protein is insulin and the diabetes is Type I diabetes. Further optionally, the diabetes is Type II diabetes.
In the drawings and tables,
Supplementary
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Supplementary Table 1. Size, PdI, and derived count rate (DCR) of insulin core particles (See.
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Supplementary Table 2. Average loading capacity and L-arg content in insulin- and exenatide-SiNP.
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Supplementary Table 3. Diameter and zeta potential mean values for particle reproducibility.
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Supplementary Table 4. Average hydrodynamic diameters of insulin-SiNPs at key stages of synthesis.
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Supplementary Table 5. Hydrodynamic diameter as denoted by Z Average and Number Mean of samples made by two operators.
Supplementary
Supplementary Table 6. Mean hydrodynamic diameter of five exenatide core particle and exenatide-SiNP batches.
Supplementary Table 7. Comparison of the content of five independent washed insulin-SiNP particle batches synthesised side-by-side.
Supplementary Table 8. Comparison of the mean sizes of the particle suspensions: data obtained at both UCD and Sanofi (Montpellier). One batch was divided and analysed.
Supplementary
Supplementary Table 9. Comparison of the mean zeta potentials of the exenatide-SiNP suspensions obtained at UCD and Sanofi.
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Supplementary Table 10. Comparison of the mean zeta potentials of the exenatide-SiNP suspensions obtained at UCD and Sanofi.
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Supplementary Table 11. Mean hydrodynamic diameter of insulin core particles made in reactions of varying volume.
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Supplementary Table 12. Characterisation and loading of SiNP. 25° C. with fluorescence and HPLC, Concentration was 100 μg/ml under gentle stirring, SIF without pancreatin, Data shown as mean±SD (n=3).
Supplementary
Supplementary
Synthesis of SiNPs Comprised of a Protein Core Crosslinked with Silica
The aim was to develop a silica-coated core which would enable oral peptide delivery. Initially core particle formation was optimised using insulin glulisine (insulin, 5.1 kDa, Accession Number: DB01309), as a model peptide. Protein cores were also made using exenatide (4.8 kDa, Accession Number: P26349and bovine serum albumin (BSA, 69 kDa, Accession Number: P02769) as payloads. Synthesis comprised four stages, Step 1: peptide/protein dispersion, Step 2: L-arg addition, Step 3: ZnCl2 addition, and Step 4: silica crosslinking (
Synthesis of Insulin Core Nanoparticles
The pH, the type of amino acid, and insulin concentration affected formation. Particles did not form if the pH was <7, while large aggregates formed when pH>10 (
An insulin concentration of >1 mg/mL led to visible uncontrolled particle aggregation confirmed by DLS (
Synthesis of Exenatide Core Particles
Though some of the factors impacting insulin core particle formation also applied to exenatide, there were differences. In contrast to insulin, exenatide is soluble in water and did not need pre-dispersion in HCl and could be used at a higher initial concentration of 3 mg/mL. Similar to insulin, a pH range of 7-10 was required for particle synthesis (Supplementary
Synthesis of BSA Core Particles and Initial Summary
BSA was also used as a model for protein-based particles41. We synthesised BSA core particles with a mean diameter of 430 nm using conditions and kinetics similar to those used for exenatide core particles (Supplementary
Crosslinking Core Particles with Silica to Make SiNPs
For oral administration, core particles need to be stabilized to protect payload structure and functionality and to allow controlled release. Silica is known to condense around particulates42 and therefore we hypothesised that silica crosslinking might be suitable for coating and stabilizing core particles entrapping payload.
Properties of Insulin- and Exenatide-Entrapped SiNPs
Properties were studied after core particle formation, after crosslinking (SiNPs, pre-centrifugation wash step) and after purification (SiNPs, i.e. washed twice by centrifugation and resuspension). Silica coating of core particles was confirmed by TEM, whereas uncoated particles fell apart during TEM preparation and had a diameter twice of that measured by DLS (Supplementary
Loading Capacity of Insulin- and Exenatide-SiNPs.
HPLC and NMR were used to determine SiNP loading capacity, while elemental analysis was used in some studies for confirmation. Insulin-SiNPs and exenatide-SiNPs contained >40% of protein by weight depending on synthesis conditions (Supplementary Table 2). The former had a loading capacity of 59%, and the latter had a loading capacity of 41% (Table 1). Insulin-SiNPs and exenatide-SiNPs both had loading efficiency of ˜50%, as determined by NMR, HPLC, Micro Bicinchoninic Acid (BCA) protein assay, circular dichroism (CD), and protein fluorescence labelling (see Supplementary files for methods).
Particle Behaviour in Complex Conditions
The behaviour of insulin-SiNPs and exenatide-SiNPs in biologically-relevant buffers depended on media pH and salinity. Both constructs aggregated in PBS, while remaining colloidally-stable in simulated small intestinal fluid (SIF) at pH 6.3 (Supplementary
Bioactivity of Released Insulin from SiNPs Following Sub-Cutaneous Administration to Rats
Bioactivity of SiNP-released insulin was confirmed by sub-cutaneous (s.c.) injection of the released material from washed SiNP to normal rats. A reduction in blood glucose was observed after injection of SiNP-released insulin at a dose of 1 IU/kg, which was comparable to that seen upon injection of free insulin solution at the same dose (
Efficacy of Insulin-SiNPs Following Rat Jejunal Instillations
Insulin-SiNPs were instilled into rat jejunal loops and resulting blood glucose changes were measured in response to a dose of 50 IU/kg. Insulin-SiNPs were assessed at most stages of synthesis, including insulin-arginine solution, as well as unwashed and washed insulin-SiNPs. This also included the supernatant from the insulin-SiNP wash to account for any changes due to the protein aggregation step (
To investigate this further, we mixed washed insulin-SiNPs with both a known permeation enhancer for oral peptides, sodium caprate (C10)43 and separately, L-arg. Both resulted in a reduction of blood glucose comparable to that of s.c.-injected insulin and free insulin mixed with C10 (
The exenatide-SiNPs were also assessed an oral glucose challenge mouse model following oral delivery at a 1 mg/kg dose. Exenatide (10 μg/kg) abolished the challenge-induced blood glucose excursion following s.c. administration, whereas the effect of the orally-administered unwashed- and washed exenatide-SiNPs was modest (Supplementary
Discussion
The current study demonstrated the synthesis, characterisation, and initial proof-of-concept in rodent model for an oral peptide nanoparticle construct based on a core of payload and the excipients, L-arg and ZnCl2, stabilized and coated with a silica shell. Silica presents a matrix which maintains particle structure in the presence of acidic pH, and consequently, intestinal enzymes, but which then dissolves as the pH rises in the small intestine34. Stable crosslinked particles were formed, with properties which depended on the payload. Additionally, we examined over 20 factors in the formation of the core particles and final SiNP constructs. Notable features of the SiNP include high peptide loading capacity, an advantage over many nanoparticle prototypes, even when they have a high association efficiency, e.g.44. Without this, too much payload will be required for a practical oral formulation. The insulin in SiNP was released at a pH value present in the small intestine and was structurally intact. Both insulin- and exenatide-SiNPs were biologically functional.
We have studied the synthesis, physicochemical and in vivo reproducibility of our constructs in an attempt to adhere to recent recommendations on nanoparticle characterisation45. The reproducibility of the synthesis was thoroughly investigated across many batches (Supplementary files). Synthesis with insulin has been carried out by five researchers, some with minimal training. To briefly summarize, the variability in measuring the size expressed as relative SD of a single insulin core particle batch in DLS was ˜3%. Comparison of 5 batches made in the same study yielded a size variability for the insulin core particle of ˜11%, the pre-wash insulin-SiNPs of ˜28% and the washed insulin-SiNPs of ˜11% (Supplementary
Bioactivity of both the insulin released from insulin-SiNPs and that of the insulin-entrapped in SiNPs was preserved. The former was done by comparing released- and free insulin injected by the s.c. route to rats, while the latter was achieved by assessing the blood glucose reduction of both unwashed insulin-SiNP and washed insulin-SiNPs admixed with either L-arg or C10 in the rat jejunal instillation model. Furthermore, the in vivo results for instilled insulin-SiNP were reproducible across multiple batches. Finally, in anticipation of translation to large animal studies, synthesis scalability and storage through lyophilisation and reconstitution were established (Supplementary
The final washed insulin-SiNPs required permeation enhancement in order to reduce plasma glucose to levels comparable with that induced by the s.c.-injected insulin, albeit at 50-fold higher doses. This suggests that, in addition to established mechanisms for L-arg of stabilising insulin through promotion of monomer formation46, as well as prevention of protein aggregation in liquid formulations47, an additional role of L-arg identified here is to act as a biocompatible permeability enhancer in the SiNP. While L-arg was recently discovered to be an intestinal permeation enhancer when ad-mixed with an insulin solution and administered to rat ileal loops in vivo37, to our knowledge this is the first example of its capacity to perform an equivalent role as part of an insulin nanoparticle construct. It is possible to supplement L-arg during lyophilisation, which may both further stabilize the SiNPs and augment permeation enhancement for an optimised formulation.
It is often overlooked that most other nanoparticle constructs also include excipients, but their precise roles have not been clarified. Indeed, in one of the first prototypes used for oral insulin in the 1980s, the bile salt, sodium deoxycholate, and poloxamer 188 were included in polyalkycyanoacrylate nanoparticles and these were effective in diabetic rats48. More recently, a nanocapsule construct also intended for oral insulin comprised the known permeation enhancers, protamine, and sodium glycocholate20, yielding plasma glucose reductions upon intestinal instillation to rats. A third, albeit less efficacious, example was a polyethylene glycol (PEG)-polyglutamic acid polymeric nanocapsule entrapping insulin attached to a cell penetrating peptide, octa-arginine, in turn conjugated to cholesterol or the medium chain fatty acid enhancer, lauric acid19. Finally, the excipient and known enhancer, Tween®-80,49 was included in the oily core of a polyarginine-coated nanocapsule designed to deliver salmon calcitonin across Caco-2 monolayers, primarily taking advantage of poly-arginine's capacity to act as a cell penetrating peptide50. In each of these examples, the excipients preserved peptide stability against pancreatic enzymes, but demonstration of permeation enhancement by them was never proven, nor a mechanism suggested.
Evidence that the majority of these and similar nanoparticles are internalised to a great extent by intestinal epithelia either in vitro or in vivo is equivocal51, 52; many prototypes seem to get stuck in intestinal mucus, while those with hydrophilic coatings may permeate mucus to reach the epithelial wall53. A reasonable conclusion therefore is, once the particle negotiates the mucus, there is a permeation enhancement effect from the L-arg excipient when co-released with the peptide in high concentration close to the epithelium. In the current study, unwashed insulin-SiNP were effective in rat jejunal instillations, while the washed particles were largely ineffective. This would suggest that in the unwashed state, insulin and L-arg are both concentrated in the particle core and are weakly associated with the silica shell, with some also free in solution. This was confirmed by NMR. It is the latter two sources that are removed from the construct upon washing by centrifugation, as suggested by the NMR and CD results (Supplementary
Conclusions
Insulin-, exenatide- and BSA-SiNPs were synthesised using a simple scalable process with potential for oral delivery. We elucidated the fundamental principles of core particle formation and the physicochemical properties of the final entrapped SiNP products, as well as ensuring synthesis and characterisation reproducibility. Prototype batches were made reproducibly and were characterised in accordance with recent recommendations. Attractive features were the high payload loading as well as high proportion of insulin release at small intestinal pH values. The insulin and exenatide-SiNPs were bioactive following intestinal instillation and oral gavage to rats and mice respectively. The mechanism of action for the insulin-SiNP relies not on epithelial particle uptake, but on the co-entrapped excipient, L-arg, acting as a PE to enable the particle-released payload to cross the epithelium. Though the work relates to three payloads, we expect it to be applicable to other peptides and proteins. It is also expected that nanoparticle constructs with a high loading capacity may be useful for applications beyond oral peptide and protein delivery, including local delivery to arthritic joints54. The use of silica as a stabilizing matrix in multiple formats may also be further enhanced by functionalisation for targeted delivery to diseased tissue receptors55.
Supplementary Notes
Synthesis of Core Particles
1H NMR to Assess Amino Acid-Insulin Interactions
Liquid 1H NMR was also used to assess the role of the amino acid in insulin particle formation. Changes in the signal line-shape and integration with variation of the amino acid and its integration relative to insulin at several steps during ZnCl2 addition were analysed. Such shifts in the NMR signal chemical shift (mode value) or full width half-maximum (FWHM) are attributed to changes in the molecule environment or its mobility1.
Relating to Changes in Insulin Signal:
Relating to Changes in Amino Acid Signal:
The interactions between insulin and the selected amino acid is complex. Of the four amino acids studied, L-arg appears had the most interesting behaviour. Changes in both the insulin and L-arg signal were continuous with ZnCl2 addition, as opposed to occurring only at particle formation. This may suggest the formation of multimers of protein prior to reaching the critical ZnCl2 concentration. Due to the similarities between the molecular structure of L-arg and L-lys and the difference in the insulin behaviour, it is likely that the specific behaviour was linked to the amine-rich side chain.
The Effect of L-arg and ZnCl2 Concentration on Insulin Core Particle Formation
There was a complex relationship between L-arg and ZnCl2 dependent on solution pH. Particles did not form below pH 6.5 regardless of solution salinity, this was typically the case when the L-arg concentration was <2 mg/mL. When the pH was in the “goldilocks” zone, between ˜6.5 and ˜9.5, the ZnCl2 concentration required to form optimised insulin particles and insulin-SiNPs depended upon the L-arg concentration. A measurable change in the DLS signal (indicative of insulin association) was observed with 0.1 mM ZnCl2 when the L-arg concentration was 2 mg/mL, ˜0.3 when it was 3 mg/mL and 0.4 when it was 4 mg/mL (
Other Factors
Several additional factors were observed to affect insulin particle synthesis.
(i) with all other factors being equal, more ZnCl2 was required for particle formation to occur if the HCl concentration used during Step 1 was low; (ii) Impurities, especially other salts, impacted upon the required ZnCl2 concentration and stability of the final particles; (iii) the speed of the ZnCl2 addition as well as homogenization may lead to the formation of regions with high local ion concentration resulting in larger insulin particle diameter and/or bimodal distributions, especially with synthesis scale up (discussed below).
Insulin-SiNP Synthesis Using an Insulin: L-Arg Mass Ratio of 1:2
While using an insulin:L-arg molar ratio of 1:99 was preferable for synthesis reproducibility, it was also possible to make particles using a ratio of 1:66 with no further alterations provided there was a fine control over the synthesis conditions. Indeed, insulin core particles and resulting insulin-SiNPs made this way are typically smaller: ˜220 nm and 260 nm in diameter, respectively (Supplementary
TEOS Additions
Several factors impacted the loading capacity, size and morphology of the final SiNP. Specifically: (i) pH and stirring, (ii) rate of addition, (iii) total volume of TEOS/mL of particles added and (iv) reaction time. The first two affect the rate of TEOS hydrolysis, which in turn influences monomer silicic acid monomer generation and also the crosslinking of insulin- and exenatide-SiNPs2. We found that slow monomer generation was more beneficial to protein crosslinking, as it leads to more uniform particles with higher loading capacity and, in the case of insulin-SiNPs, a more definitive core shell morphology.
TEOS Hydrolysis Rationale
Silica forms in water spontaneously from TEOS in these conditions above a critical concentration2. It would be energetically preferable for silicic acid molecules to remain in the particle. Thus, if silicic acid concentration is kept below a critical value, its molecules will preferentially crosslink protein particles rather than form a separate silica phase. Morphology and reaction rate were controlled by controlling TEOS hydrolysis at a constant volume/mL. Hydrolysis rate was controlled by the size of the TEOS/water interface, temperature and a basic pH of 10. The images above show how the unwashed sample changes with increasing TEOS hydrolysis (Supplementary
Volume of TEOS Influences Particle Morphology
A different volume of TEOS was added to each of insulin, exenatide and BSA core particles. The dispersion was incubated for ˜24 and ˜48 h at ˜20° C. and the resulting particles were measured by DLS and TEM before and after purification. The insulin-SiNPs had a pronounced core-shell, whereas exenatide- and BSA-SiNPs were more uniform. Increasing the TEOS added to insulin particles from 0.5 μL per mL to 2 μL per mL increased particle size in a volume-independent manner over the volume range (Supplementary
Loading Capacity and Loading Efficiency
Loading Capacity
Several approaches were applied to measure the chemical composition of the SiNP. Both direct- and indirect measurements of the particle peptide content were attempted by HPLC, NMR, IR, elemental analysis, SDS-PAGE, CD, and protein fluorescence labelling. Since dispersing exenatide in water did not require HCl, initial methodology was established using Exenatide-SiNP.
Directly measuring the protein content of particles requires particle dissolution and complex sample preparation. Methodologies were separated in several categories based on best outcome requirements: (i) those for which protein structure is not essential for recognition (pH>10), i.e. NMR and HPLC; (ii) those for which at least the primary protein structure should be maintained: IR, SDS-PAGE, CD and micro BCA and (iii) methods which are not structure-sensitive: elemental analysis and protein fluorescent labelling. A detailed view of conditions for silica dissolution can be found3, 4. Complete dissolution of the SiNP proved difficult, and as a result, IR, SDS-PAGE, CD and micro BCA were not applicable. Neither was it possible to measure the chemical composition using fluorescence labelling, NMR, HPLC, and elemental analysis. Of those, fluorescently-labelling the protein prior to particle formation may have unintended effects on the process and leading to possible changes in the composition of the final dispersion or the functionality of the protein. Furthermore, it introduces several additional steps which increases the time of each reaction and is not practical when used for insulin. Elemental analysis is destructive and requires >1 mg of sample. Both were used sparingly. On the other hand, NMR and HPLC, while destructive in this context, require less sample (≈0.5 mg), can be used in tandem, and were chosen for batch composition studies. Supernatant content could be measured by most of the above methods and was used to estimate loading efficiency.
Loading Efficiency
Measuring the peptide concentrations in the supernatants was possible using most methods except for SDS-PAGE and IR, where the signal-to-noise ratio was too low, and for elemental analysis where there were high sample requirements. The capacity of NMR to simultaneously and quantitatively measure distinct species in native conditions makes it a powerful characterisation tool. Similar to direct measurement, fluorescence labelling of the protein allows for a rapid measurement of the loading efficiency, but the impracticality of the additional steps and possible influence on the reaction made it inconvenient for frequent use. CD and BCA were straightforward to use, but were problematic in distinguishing between insulin and L-arg, as both would be detectable.
Reproducibility
Synthesis Reproducibility
Synthesis reproducibility of insulin-entrapped particles in terms of physicochemical properties was measured in three ways: by comparing five side-by-side batches by a single researcher made in a single study, using a side-by-side comparison of batches synthesised by two researchers, and by an evaluation of batches made by the same researcher using the same protocol. Studying the variability between five scientific replicates made side-by-side on the same day was used to estimate variability in the synthesis. This variability depended on the stage of the synthesis: insulin core particle batches made on the same day had a Z average hydrodynamic diameter of 329 nm with a variability of 11% (37 nm). This was above the measurement variability (typically ˜10 nm or 3%). The same particles had a number mean hydrodynamic diameter of 236 nm with a variability of 11% (Supplementary
Examining variability of the synthesis was further assessed. The average hydrodynamic diameter measured for insulin core particles was 290 nm, with a relative SD of 18% (N=34). For the insulin-SiNPs, it was 488 nm with a variability of 38% (N=35), and for washed insulin-SiNPs, it was 451 nm with a variability of 15% (N=24) (Supplementary
A similar side-by-side experiment was used to establish the reproducibility of particles. The average hydrodynamic diameter of five scientific repeats was 126 nm±14 nm (11%) for exenatide core particles and 133±13 nm (9%) for washed exenatide-SiNPs (Supplementary
Characterisation Reproducibility
We compared the physicochemical properties of an exenatide-SiNP batch, divided between UCD and Sanofi (Montpellier). Particles were initially synthesised and characterised at UCD, and the remainder of the batch was shipped to Montpellier in a liquid suspension. Their capacity to reduce plasma glucose was tested using a glucose challenge in a mouse model.
Comparing Particle Size
Exenatide-SiNPs were synthesised and characterised at UCD after which the liquid dispersion was sent to Sanofi for repeat analysis using the same protocol. Exenatide-SiNPs unwashed, and after one and two washing steps were studied. Size of the particles was measured through DLS, Z average, intensity and number mean. Variability between particle diameter between the two institutions was less than 5% in all cases (Supplementary Table 8). Particle size distribution obtained at both institutions were comparable (Supplementary
Comparing Zeta Potential
At both labs, zeta potential was measured using a Malvern Zetasizer ZS series with Zeta potential cuvettes (model number: DTS1070). Measurements at UCD were done in a 1/100 dilution of PBS (I=1.4×10−3 M), while those at Sanofi were done in deionised water. All samples had a negative zeta potential, however, values measured at Sanofi were closer to neutral (Supplementary Table 9). This can be attributed to the lower medium conductivity in Sanofi measurements. Zeta potential distributions between both institutions were comparable (Supplementary
Comparing Exenatide-SiNP Loading Capacity
In both labs particles were dissolved in basic conditions and the exenatide content was measured using a C18 column in reverse phase HPLC. The mobile phase used was 69% water, 30% acetonitrile and 1% THF. Results were compared to a calibration curve. Exenatide content for the exenatide-SiNPs after one and two washing cycles was less than 10% (Supplementary Table 10). This is similar to what was obtained between batches.
Further Considerations
Reaction Scale-Up
Though the reaction was found to be scalable, several factors were important for the quality of the final product. (i) Reaction volume homogeneity: i.e. the appearance of concentration gradients within the reaction leading to a broad or bi-modal distributed particle population. This problem was addressed by appropriate homogenization and reaction vessel choice; (ii) Regions with high local ZnCl2 concentration:ZnCl2 addition rate and homogenization were important at higher volumes. Thus, the addition rate was reduced and an appropriate homogenization strategy adopted; (iii) Regions with high local TEOS concentration: both the particle crosslinking/coating and the formation of a secondary silica phase were dependent upon the rate of TEOS hydrolysis, which relates to reaction conditions and TEOS concentration. In high volume reactions, local TEOS maxima can appear due to reagent point addition. Similar to the two previous considerations, the problem can be resolved with adequate homogenization and a reduction of the TEOS addition rate using microfluidization. No significant size variability was detected when these considerations were taken into account (Supplementary
Using NMR to Monitor the Reaction Mechanism
We further used NMR to monitor the insulin-SiNP synthesis, conducted in D2O. There was little change in the L-arg and insulin NMR signals after they were mixed, indicating weak association (Supplementary
Long Term Sample Storage: Lyophilization
An insulin-SiNP batch was separated in seven aliquots, one was left in water, while 2, 4 and 6% of sucrose or trehalose were added to six aliquots. The resulting dispersions were further separated in two sets. One set (1st) was left at room temperature (˜20° C.), while the other (2nd) was frozen at −80° C. overnight and lyophilised over 36 h. Samples of the 2nd set were reconstituted in water and dispersion was compared to their counterparts from the 1st set. We found that even at the lowest concentration of either sugar, the dispersions of the 1st and 2nd set were similar, while samples aggregated in the absence of sugar (Supplementary
Materials and Methods
Materials
Methods
Insulin Core Particle Synthesis Using an Insulin:L-arg Molar Ratio of 1:99
Preparing the Insulin Suspension.
The required mass of insulin (insulin glulisine, Sanofi) was weighed in a clean, dry plastic container. HPLC grade water was added so that the final concentration was 2 mg/mL. Insulin did not dissolve, but formed a white. 10 μL/mL 1 M HCl was added to the heterogeneous mixture which was further vortexed. A transparent colourless solution was obtained, with no floating particles.
Core Particle Synthesis.
6 mg/mL L-arg was added to the insulin solution in a 1:1 volumetric ratio so that the final concentrations were 1 mg/mL insulin and 3 mg/mL L-arg. The solution was vortexed for a few seconds and left for several minutes to equilibrate. The pH of the solution was >8.5. This is a DLS point. ZnCl2 was kept as a stock solution at a concentration of 200 mM in HPLC grade water following sonication. While the insulin+L-arg was equilibrating, the ZnCl2 stock solution was diluted 20 fold in HPLC grade water to a final concentration of 10 mM. Sonication/vortexing is recommended to ensure the homogenization of stock and diluted solutions. 3×30 μL/mL aliquots of the diluted ZnCl2 solution were added to the insulin+L-arg solution with homogenization between each addition. The solution was colourless and clear. Additional ZnCl2 was added in 5 μL/mL increments with agitation until a white, turbid suspension was formed. In a typical synthesis, 70 μL ZnCl2 was required to cause aggregation. The solution, at this stage was turbid but transparent when held up to light. This is a DLS point. The process was pH-dependent, as insulin did not aggregate if the pH range was not 8-10.
TEOS Addition and Particle Purification.
The core particles were left for at least 1 h to equilibrate before adding 1 μL/mL TEOS to the solution. During that time continual agitation was provided. If the reaction volume is >10 mL, slow spinning ensured homogeneity. If the reaction volume was high, TEOS was added in several steps to prevent generation of monomer in the solution and thereby a secondary silica phase. The suspension was left for at least 24-48 h. This is a DLS point. To wash, particles were distributed in 2 mL Eppendorf tubes and spun at 14 000 rpm for 30 min. The supernatant was removed and the particles were dispersed in water by gently pipetting them up and down in the Eppendrof, flicking with a finger and/or vortexing. This is a DLS point. Insulin-SiNPs were washed twice by centrifugation.
Insulin Core Particle Synthesis Using an Insulin:L-arg Molar Ratio of 1:66
Deionized water (HPLC-grade) was syringe-filtered through 0.2 μm filter membranes. An aliquot of this water was acidified to pH 2 with HCl. Concentrated L-arg (0.14 M) and ZnCl2 (0.08 M) stock solutions were prepared. These solutions were sonicated for 10 min to ensure complete dissolution. Insulin was weighed out on a microbalance such that each sample tube contained 1 mg. L-arg and ZnCl2 solutions were diluted to 0.023 M and 0.011 M respectively. Insulin was solubilized in HCl (0.01 M) and vortexed for 30 s (15 Hz). Insulin stock (500 μL) was added to an empty microcentrifuge at a concentration of 1 mg/mL and shaken at 25° C., 500 RPM. L-arg (500 μL) was then added to this solution (c=2 mg/mL) while shaking at 900 RPM. Subsequently, an aliquot of ZnCl2 solution was added to the sample tube such that the final concentrations in the sample tube were:
Upon addition of ZnCl2, a cloudy colloidal suspension was observed, which may be checked by shining a laser pointer through the sample tube. If colloids formed, a scattered beam was observed through the suspension. If no scattering occurred, peptide nanoparticles were unlikely to be present. If a colloidal suspension did not form, a further aliquot addition of ZnCl2 may be added to the sample tube. Insulin core nanoparticles formed for molar ratios of ZnCl2:L-arg from 1:21 down to 1:14. At low ZnCl2 concentrations (molar ratios below 1:21), peptide nanoparticles formed, but hexamers were also likely present. Larger aggregates formed at high ZnCl2 concentrations, thus, to improve reproducibility of the final peptide nanoparticle size, an optimal molar ratio of 1:21 was chosen which returned a mean hydrodynamic nanoparticle size of 220 nm. Samples were left shaking for 6 h after ZnCl2 addition to ensure the sample had equilibrated. Samples were left overnight before TEOS addition. A small volume of TEOS (2 μL) is added (t=17 h) to each sample tube at 900 RPM; temp=37° C. Samples were left for 24 h to ensure complete hydrolysis of TEOS. Insulin-SiNP were washed using a microcentrifuge at 14000 RPM (16,873 g) for 15 min. The supernatant was carefully removed with a pipette and the pellet was re-suspended in deionized water.
Samples were checked using DLS at two stages: after preparation of peptide nanoparticles and after TEOS addition. An increase in the hydrodynamic size was observed after addition of TEOS indicating the formation of a silica shell on the outer peptide nanoparticle surface. After washing via centrifugation and resuspension, TEM samples were prepared by depositing <5 μL of suspension on a TEM grid. The sample was then covered and allowed to dry in ambient conditions. Peptide secondary structure conformation may be assessed at any stage of the process by depositing solution/suspension into a quartz cuvette and performing a circular dichroism measurement using a spectropolarimeter (180-300 nm).
Exenatide-SiNP Synthesis
Exenatide was solvated in HPLC grade water at a concentration of 3 mg/mL. The mixture was homogenized using inversion and vortexing for 30 s and left to equilibrate for 1 min. 166 μL/mL of 24 or 45 mg/mL L-arg was added, depending on required final size. The former results in exenatide core particles with a mean dimeter of 50-100 nm, while the latter yields ˜200 nm ones. The solution was homogenized again using vortexing and left for further 5 min to equilibrate. The final solution was a clear, colourless liquid with a pH of around 9. 100 mM ZnCl2 was added slowly to the exenatide+L-arg solution using 9×10 μL/mL aliquots once every 10 min. The resulting exenatide+L-arg+ZnCl2 solution was a clear, colourless liquid. It was left stationary or, if the volume of the reaction was large, slowly spinning at 20-25° C. for 24 or 48 h, depending on particle size for the particle to grow.
TEOS (2 μL/mL) was added to the synthesized exenatide core particles. The reaction was left either stationary or slowly spinning for further 24-48 h. This is a DLS point. To wash, particles were distributed in 2 mL Eppendorf tubes and spun at 14 000 rpm for 30 min. The supernatant was removed and the particles were redisposed in water by gently pipetting them up and down in the Eppendorf, flicking with a finger and/or vortexing. This is a DLS point. The final suspension was a semi-turbid, white liquid. The final particle size was characterised as for insulin-SiNP. Typical exenatide core particle and washed exenatide-SiNPs had a Z-Average between 100-200 nm with a PdI<0.2. Encapsulation efficiency was >90% (by NMR) and ≈50% (by NMR and HPLC), respectively. Exenatide core particles had a loading capacity of ≈50% (by NMR, HPLC and EA).
BSA-SiNP Synthesis
BSA was solvated in HPLC grade water at a concentration of 3.1 mg/mL. The mixture was homogenized using inversion and vortexing for 30 s and left to equilibrate for a further minute. 250 μL/mL of 24 mg/mL. L-arg was then added and the solution homogenized again using vortexing and left for further 5 min to equilibrate. The final solution was a clear, colourless liquid with a pH of around 9. 200 mM ZnCl2 was added slowly to the BSA+L-arg solution as 2×20 μL/mL and 1×5 μL/mL aliquots once every 10 min. The time between additions was measured using DLS. The mixed solution was a clear, colourless liquid and appeared largely unchanged. It was left stationary or, if the volume of the reaction was large, slowly spinning at 20-25° C. for 4 hours for the particle to grow. TEOS was added in a volume of 2 μL/mL to the core particles. The reaction was left slowly spinning for further 72 h. To wash, the particles were distributed in 2 mL Eppendorf tubes and spun at 14 000 rpm for 30 min. The supernatant was removed as described as above. The final particle size was characterised as for insulin-SiNP.
Storage
All particles were stored at 4° C. for up to a month.
Characterisation Methods
DLS
The volume of solution was >0.5 mL in water or aqueous buffer all cases.
Proteins: Proteins were dispersed in water or appropriate media at a concentration of ≥1 mg/mL.
Protein core particles: The pure protein particles were carefully transferred into the cuvette without further dilution.
SiNP, unwashed or washed: These particles were either measured as synthesized or diluted 1/100 in the appropriate media.
TEM
Particles (˜1 mg/mL) were diluted 1/50 in water or an appropriate buffer. 5-10 μL of the vortexed suspension were transferred to a 400 mesh copper grid and left to dry for >1 h. A standard Techni 12 instrument set to 120 keV was used in all cases. TEM data was analysed manually using ImageJ®.
Circular Dichroism
A Jasco J-810 was used for all measurements. Parameters used were as follows:
Insulin, L-arg solutions, mixtures and insulin core and SiNPs were measured at appropriate concentration in an ultra-low volume (1=1 mm) cuvette and at appropriate concentrations. Comparison was made using the intensity of the insulin peak at 220 nm and the L-arg peak at 210 nm. Results were compared to standard curves. The high tension (HT) in all cases was below 800 V unless otherwise specified.
HPLC
A C18 column and a 69% water, 30% acetonitrile and 1% THF mobile phase were optimal for measuring both free exenatide and insulin, as well as peptides released from particles. Solutions used had a pH<10.
Measuring Free Protein.
Water was run first for 20 min at a rate of 0.5 mL/min to let the column equilibrate with the mobile phase and then again for 10 min at a rate of 1 mg/mL. Samples were run at 1 mg/mL flow rate in the above mentioned conditions.
Measuring the Peptide/Protein Loading Capacity of SiNPs.
Typically, particles were dissolved using 0.2 M NaOH for >3 h at 37° C. Then the pH was adjusted to ˜8 using 1M HCl and the samples were transferred to a standard HPLC vial. Water was run as a sample for 30 minutes prior to sample measurement. Samples were run within 2 h of the pH adjustment to prevent silica reconstitution. The final result was compared to a standard curve made of the protein under study at the same conditions. Typically it takes the sample<10 min to elute out of the column at a flow of 1 mL/min.
Calibration Curve for Estimating the Loading Capacity of Protein-SiNPs.
The protein was treated as a sample. It was dispersed in a predetermined concentration and an amount of HCl (10 μL/mL) was added in the case of Insulin. NaOH was added so that its final concentration was 0.2 M. The proteins were left for at least 3 h at 37° C. After which the pH was brought down <10 using HCl, as measured by pH paper. The as obtained protein solution was diluted and measured the same way as described above.
NMR
Preparing a Calibration Curve
Protein concentration was measured through a calibration curve made in appropriate conditions. Calibration for free protein was carried out either by dissolving it in D2O or, in the case of insulin, D2O/HCl (pH 2), after which the pH was fixed to 7 in all cases. Calibration for determining protein concentration after dissolution was made by incubating the free protein in 0.2 M NaOH at 37° C. for several hours and measuring the solution without further pH adjustments.
Measuring Unwashed- or Washed Particle Dispersions.
Insulin-L-arg mixtures, protein core particles or protein-SiNPs were either made in D2O or spun into D2O twice without any intentional dilution.
Measuring Loading Capacity.
Typically a known sample volume was dried in a pre-weighed Eppendorf tube (using a 6 place balance). The mass of the dry nanoparticles was measured and subsequently dissolved in 1 mL of 0.2 M NaOH, 1 mM DSS solution for >5 h at 37° C.
NMR Settings.
The peak at 0 ppm of (4,4-dimethyl-4-silapentane-1-sulfonic acid) at a concentration of 1 mM was used as an internal standard for all measurements. They were done with 16 scans with a relaxation time of 25 sec in a Varian 400 and 600 MHz instrument using 600 MHz NMR tubes.
Data Analysis.
Data was analysed using the automatic and manual peak fitting in the MestReNova V11 software.
In Vivo Methods
Rat Intestinal Instillations
Male Wistar rats (UCD Biomedical Facility and Charles River, UK, 7-10 weeks of age, bodyweight 300-400 g) were housed under environmentally-controlled conditions of humidity and a 12:12 h light/dark cycle. Animals had access to filtered water and standard laboratory chow ad lib. All experiments were conducted in accordance with Health Products Regulatory Agency project authorisation AE18982/P036 and UCD Animal Research Ethics Committee protocol number 13-40. All procedures were performed under anaesthesia. Before anaesthesia, animals were fasted overnight with free access to filtered water. Isoflurane (Iso-Vet, 1000 mg/g isoflurane liquid for inhalation, Piramal Healthcare, UK) was used for induction (5 L/min mixed with 4 L/min O2) and maintenance of anaesthesia (2 L/min mixed with 2 L/min O2). The in vivo bioactivity of released insulin was investigated. Insulin solution or insulin released from SiNPs was injected (s.c) at 1 IU/kg using a 26 G needle. PBS was also administered as a control. In situ instillations were performed as previously described5 with minor modifications. In brief, midline laparotomy was performed and an intestinal loop (4-6 cm) was created using size 4 braided silk suture (Mersilk®, Ethicon Ltd., Uk). Care was taken to avoid obstructing any vasculature. The abdominal cavity was closed using a skin stapler (AZ Manipler 35W, Braun). PBS (control) or treatments (Insulin, insulin-SiNP (50 IU/kg insulin) were instilled using a 30 G needle. In the case of the insulin-SiNP, an equal volume of PBS was administered into the loop prior to the NP solution to aid dispersion. Glucose levels were measured with blood obtained from the tail vein every 20 min (T0-T120) using a glucometer (Accu-chek Aviva, Roche). Animals were euthanised by intracardiac injection of 0.5 mL of pentobarbital sodium (EUTHATAL™, 200 mg/ml, Merial Animal Health Ltd., UK) using a 21 G needle. After euthanasia, the intestinal loop was placed in 10% formalin and subsequently embedded in paraffin wax. 5 μm tissue sections were cut on a microtome (Leitz 1512; GMI, USA), mounted on adhesive coated slides, stained with haematoxylin/eosin (H&E) and Alcian blue and examined under light microscopy (Nikon Labphoto; Nikon, Japan).
Oral Administration of Exenatide-SiNPs to Mice
Female C57BL/6 mice with a body weight of 19-25 g were used. Mice were randomly assigned to treatment groups and deprived of food for 6 h prior to study start but with tap water ad libitum. Controls were treated orally with 0.1M TRIS buffer of pH 8.0 (n=12). Test groups were treated with exenatide-SiNPs (p. o., n=10) or exenatide (s.c., n=10) respective doses of 1 mg/kg and 10 μg/kg, respectively. An oral glucose tolerance test (OGTT) using 2 g/kg p.o. glucose was initiated 0.5 h after treatment. Blood glucose was determined between 0 h (=time point of drug treatment) and 4 h. The PK of exenatide was analysed 0.5 and 1 h after administration from K 3-EDTA-treated plasma using an exenatide ELISA (LLoQ=11.2 pg/mL).
| Number | Date | Country | Kind |
|---|---|---|---|
| 1913606.8 | Sep 2019 | GB | national |
| 1913609.2 | Sep 2019 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/076339 | 9/21/2020 | WO |
