WO2006102762A1 - Microgels reagissant au glucose - Google Patents

Microgels reagissant au glucose Download PDF

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Publication number
WO2006102762A1
WO2006102762A1 PCT/CA2006/000487 CA2006000487W WO2006102762A1 WO 2006102762 A1 WO2006102762 A1 WO 2006102762A1 CA 2006000487 W CA2006000487 W CA 2006000487W WO 2006102762 A1 WO2006102762 A1 WO 2006102762A1
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microgel
glucose
pba
microgels
functionalized
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PCT/CA2006/000487
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Todd Hoare
Robert Pelton
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Mcmaster University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F1/00—Treatment of water, waste water, or sewage
    • C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
    • C02F1/54—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities using organic material
    • C02F1/56—Macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels

Definitions

  • the present invention relates to microgels.
  • Microgels are intermediates between linear polymers and hydrogels. Like hydrogels, microgels have a covalently-crosslinked, three-dimensional structure and can swell and deswell in response to changes in the gel environment; like linear polymers, they are relatively small in size ( ⁇ 1 micron) and consequently have very high specific surface areas, respond quickly to environmental stimuli, and can be made to flow.
  • the present invention comprises a technique of grafting aminophenylboronic acid to a pre-existing carboxylated microgel via EDC coupling to make PBA-functionalized microgels.
  • the technique very well-defined carboxylated microgels with different radial and chain functional group distributions can be synthesized.
  • the PBA- functionalized microgels can exhibit controllable and predictable swelling responses.
  • the microgels can also be produced by copolymerization of a PBA-containing monomer.
  • Super-responsive microgels can be designed with a volume phase transition temperature (VPTT) just below physiological temperature in the absence of glucose and just above physiological temperature in the presence of glucose.
  • VPTT volume phase transition temperature
  • the PNIPAM thermal transition is used to magnify the PBA-glucose interaction-induced transition within the microgels to achieve higher degrees of swelling response to a particular glucose concentration.
  • the temperature at which the transition occurs can be modified by adjusting the -COOH content of the microgel, the
  • SUBSTiTUTE SHEET (RULE M) degree of conjugation, and/or the distribution of functional groups in the microgel. This permits precise tuning of the temperature and/or glucose concentration at which large responses are desired as well as allowing the switch to be effectively turned “on” and “off' simply by changing the temperature.
  • amphoteric microgel allows (1) the design of microgels which are responsive to changes in glucose concentration at physiological pH (7.4) via the incorporation of cationic groups into the microgel; (2) a net negative charge to be maintained on the surface of the PBA-grafted microgel at all physiologically-relevant pH values, significantly reducing the risk of an immune response; (3) the exploitation of the anti-polyelectrolyte behaviour of amphoteric microgels to achieve significantly higher stability at high ionic strengths, permitting glucose-induced swelling under physiological conditions (pH 7.4, 0.15M ionic strength, and 37 0 C); and (4) the synthesis of microgels which can contract in the presence of glucose as well as microgels which can switch their response between contraction and swelling according to the pH value of the medium (via regulation of the positive-negative charge balance within the microgel).
  • microgel probes can be synthesized for measuring glucose concentrations in complex mixtures.
  • the probes can be readily tuned (both in terms of the magnitude as well as the direction of the desired response) based on the temperature, pH, and salt concentration of the medium used.
  • the anionically-initiated microgels are also expected to be ideal for use in self-regulating in vivo insulin control devices given their unique ability to swell up to 70% by volume under physiological pH, temperature, and ionic strength.
  • the present invention should also permit the construction of radiolabeled biotracking devices which can bind and detect specific analytes.
  • microgels prepared can bind selectively to glucose and can be made to swell (or deswell, depending on the type of delivery envisioned) systematically with increases in the glucose concentration within the physiologically-relevant glucose concentration range
  • microgels are highly uniform and easily injectible (unlike macrogels which have previously been synthesized), such a system should be useful as a self-regulating diabetes treatment, reducing the frequency of insulin inj ections and eliminating the risk of insulin overdose and subsequent hypoglycemia.
  • PBA-containing microgels with a low surface charge can selectively bind to these carbohydrates in the cell glycocalyx and viral envelope to selectively remove such materials from complex mixtures. This would allow, for example, water to be made “safe” by using a comparatively small amount of flocculant since very little of the polymer would be "wasted” in removing inert materials (ie. clays, sands, minerals) from the water.
  • microgel-cell/virus complex from the other components of the mixture (easily accomplished with microgels via filtration or centrifugation) and subsequently recover the biological component by increasing the temperature and/or decreasing the pH.
  • the large surface area reduction achieved in thermal deswelling may sterically induce desorption; in the latter case, since glucose only binds to ionized PBA groups, lowering the pH below the pKa of the boronic acid groups (ie. from pH 7.4 to pH 6, well within the "safe" pH range for handling cells) would eliminate the affinity of PBA groups for the carbohydrates and induce desorption.
  • the microgel could also be recycled for multiple use if desired.
  • SUBSTITUTE SHEET (BULE 20) By monitoring either the particle size or the intensity of light scattered by the PBA- conjugated microgels, one can measure the concentration of glucose in solutions in situ without requiring complex separation/isolation procedures. Of particular significance, this detection can be performed in physiological ionic strength solutions using the present invention. Furthermore, by changing the temperature and/or pH of the medium, one can selectively turn the microgel detection system on or off as desired. Microgels with enhanced sensitivity over specific glucose concentration ranges can also be synthesized to provide higher resolution to detect smaller glucose concentration changes over ranges of interest. Furthermore, the glucose concentration range over which this additional sensitivity occurs can be tuned simply by changing the operating temperature of the microgel sensor.
  • microgels can be designed to both swell and deswell in the presence of glucose, persons of ordinary skill in the art using this disclosure could easily design a microgel switch which would close (swelling gel) or open (deswelling gel) as the glucose concentration is increased.
  • Figure 1 shows ratio change in hydrodynamic volume (V, Fig. 8.1 (a)) and light scattering intensity (LSI, Fig. 8.1(b)) as a function of glucose concentration for the APBA-modified microgels at pH 9 and 25 0 C (5mM ionic strength).
  • SUBSTITUTE SHEET (RULE 20) corresponding electrophoretic mobilities measured at Omg/mL (top) and lmg/mL (bottom) glucose concentrations are also shown above each microgel data in panel (a).
  • Figure 2 shows hydrodynamic versus temperature profiles for the APBA-AA-11 A5 E50 microgel in the presence of physiologically-relevant glucose concentrations at pH 9.
  • the VPTT trend shown along the x-axis illustrates to the midpoint VPTT value corresponding to the transitions at each glucose concentration.
  • Figure 3 shows volumetric (Fig. 3(a)) and light scattering intensity (Fig. 3(b)) ratios for APBA-AA-11 A5 E50 as a function of glucose concentration at different temperatures (pH 9, 5mM carbonate buffer)
  • Figure 4 shows hydrodynamic diameter versus temperature profiles for PBA-microgel conjugates prepared using microgel platforms with different functional group distributions (pH 9, 5mM carbonate buffer).
  • the filled points represent the size profile in the absence of glucose while the unfilled points show the diameters in the presence of lmg/mL glucose.
  • Figure 5 shows ratio change in hydrodynamic volume (lighter bars) and light scattering intensity (darker bars) and absolute change in electrophoretic mobility (text above/below bars) in a pH 7.4 5mM phosphate buffer for APBA-17-4 microgels prepared under different conjugation conditions when exposed to lmg/mL glucose
  • Figure 6 shows ratio change in hydrodynamic volume (light shaded bars) and light scattering intensity (dark shaded bars) and absolute change in electrophoretic mobility (text above bars) for cationic-initiated amphoteric microgels exposed to lmg/mL glucose at different pH values (5mM carbonate or phosphate buffers).
  • Figure 7 shows ratio change in hydrodynamic volume (blue bars) and light scattering intensity (purple bars) and absolute change in electrophoretic mobility (text above bars) for anionically-initiated amphoteric microgels exposed to lmg/mL glucose at different pH values (5mM carbonate or phosphate buffers).
  • Figure 8 shows size distributions of E. coli. suspensions upon the addition of the (a) AMPH(+)-17-5 A5 E50 or (b) AMPH(+)-17-5 A5 ElOO microgel upon the addition of various glucose concentrations (pH 7.4, 5OmM carbonate buffer, 25°C). Size distributions are measured 10 minutes after microgel addition.
  • Figure 9 shows size distributions of E. coli. suspensions upon the addition of the cationic-initiated amphoteric microgel APBA(+)-17-5 in the absence of glucose (pH 7.4, 5OmM carbonate buffer, 25°C). Size profiles are recorded one half hour after microgel addition.
  • Figure 10 shows size distributions of P. pastoris upon the addition of (a) APBA-22-0 A5 E20 and (b) APBA-22-0 A5 ElOO as a function of added glucose (pH 9, 5OmM carbonate buffer, 25°C). Size profiles are recorded one half hour after microgel addition.
  • Figure 11 shows size distributions of P. pastoris suspension upon the addition of microgels containing different PBA contents in the absence of glucose (pH 9, 5OmM carbonate buffer, 25 0 C).
  • Figure 12 shows cell-selective fiocculation using the anionic PBA-functionalized microgel APBA-22-0 A5 ElOO (pH 9, 5OmM carbonate buffer, 25°C): (a) E. coli. response; (b) P. pastoris response
  • Figure 13 shows effect of the carbohydrate inhibiting agent on yeast fiocculation control: (a) particle size distributions; (b) laser obscuration values as a function of fiocculation time
  • Figure 14 shows effect of salt on glucose fiocculation control: (a) particle size distributions; (b) laser obscuration values as a function of fiocculation time
  • Figure 15 shows effect of the pH on yeast flocculation control: (a) particle size distributions; (b) laser obscuration values as a function of flocculation time
  • Figure 16 shows hydrodynamic diameter and light scattering responses of a series of microgels with different total PBA contents (pH9, 25 0 C, 5mM ionic strength): (a) light scattering intensity ratio (relative to zero-glucose); (b) light scattering intensity trend expanded for the APBA-22-0 and APBA-11-0 microgels in the 0-0.2mg/mL glucose range; (c) hydrodynamic diameter ratio (relative to zero-glucose); (d) hydrodynamic diameter trend expanded for the APBA-22-0 and APBA- 11-0 microgels in the 0- 0.2mg/mL glucose range
  • Figure 17 shows electrophoretic mobility versus pH profiles for APBA(-)-28-8 A2 E20 and APBA(-)-28-8 A5 E50 in the presence and absence of 2mg/mL glucose.
  • N-isopropylacrylamide (NIPAM, 99%, Acros Organics) was purified by recrystallization from a 60:40 toluene:hexane mixture.
  • Methacrylic acid (MAA, 99%, Aldrich)
  • acrylic acid (AA, 99%, Aldrich)
  • DMAEMA N,N-dimethylamine ethyl methacrylate
  • N,N-methylene-bis- acrylamide (MBA, 99+%, Aldrich), vinylacetic acid (VAA, 97%, Aldrich), fumaric acid (FA, 99%, Aldrich), cetyltrimethylammonium bromide (CTAB, 95%, Aldrich), sodium dodecyl sulfate (SDS, 98%, Aldrich), 2,2'-azobis(2-methylpropionamindine) dihydrochloride (AMPA, 97%, Aldrich), ammonium persulfate (APS, 99%, BDH), N-3- dimethyl(aminopropyl)-N'-ethylcarbodiimide (EDC, commercial grade, Aldrich), and 3- aminophenylboronic acid (APBA, 98%, Aldrich) were all used as received.
  • Microgel Preparation Polymerizations were conducted in a 25OmL three-necked flask fitted with a condenser and a glass stirring rod with a Teflon paddle.
  • a series of anionic microgels with the same total carboxylic acid content was synthesized using four different functionalized co-monomers; the recipes used are given in Table 1.
  • the amount of functional monomer (MAA, AA, FA, or VAA) used was determined such that each end-product microgel contains the same number of moles of - COOH groups per gram of gel (as measured by conductometric titration).
  • microgels with different acrylic acid contents and two microgels with different VAA contents were synthesized; the recipes used in those preparations and the final carboxylic acid content of the microgels (as measured by conductometric titration) are given in Table 2.
  • a series of amphoteric microgels was synthesized using acrylic acid (AA) as the anionic monomer and N,N-dimethylamino ethylmethacrylate (DMAEMA) as the cationic monomer.
  • AA acrylic acid
  • DMAEMA N,N-dimethylamino ethylmethacrylate
  • Microgels were also prepared using a mixed charge system of an anionic initiator and a cationic surfactant.
  • this mixed anionic initiator/cationic surfactant system was observed to give much higher overall microgel yields (-90% compared to -50% with the cationic initiator system), improved colloidal stability, and better control over the particle size of the product microgels over a broader size range compared to the fully cationic system. This is a key finding for practical scale-up of the synthesis of these microgels.
  • Anionic- stabilized microgels tend to be more stable in complex mixtures (since most biological colloids carry a net negative charge) and anionic initiators are less susceptible to chain transfer, permitting higher yields of somewhat larger particles to be achieved.
  • SUBSTITUTE SHEET (PAJIE 26) a nitrogen purge. After 30 minutes, the initiator was dissolved in 1OmL of water and injected into the reactor. Polymerizations were carried out for 20 hours under 200RPM mixing. After cooling, all microgels were purified by at least five cycles of ultracentrifugation (Beckman model L7-55 at variable speeds according to the density of the microgel being purified), decantation and redispersion in water until the supernatant conductivity was less than 5 ⁇ S/cm. Given the high conversions observed in the mixed- charge synthesis strategy used in the preparation of the anionically-initiated microgels, dialysis could also be used for microgel purification. Microgels were lyophilized and stored dry at 4 0 C.
  • Phenylboronic acid functional groups were incorporated on to the microgels via a "graft to" approach.
  • EDC was used as a zero-length crosslinker to conjugate 3-aminophenylboronic acid (APBA) to the carboxylic acid groups contained in the base microgels. All conjugations were performed by suspending 36mg of lyophilized microgel in 3.6mL of MES buffer (pH 4.7, 2OmM ionic strength) in a scintillation vial.
  • APBA solutions were prepared at different concentrations (ranging from lmg/mL up to lOmg/mL) in the same MES buffer and 3.6mL of the desired concentration solution was added to the microgel suspension and mixed under gentle magnetic stirring at 15°C. After ⁇ 30 minutes, 1.OmL of a freshly-prepared EDC solution (again varying in concentration between 5mg/mL and 50mg/mL according to the desired degree of conjugation) was immediately added to the vials.
  • microgels referred to herein are given the code APBA-x-y Ap Eq, where x is the AA feed mole fraction, y is the DMAEMA feed mole fraction, p is the concentration of the APBA solution added (in mg/mL) and q is the concentration of EDC solution added (in mg/mL).
  • x is the AA feed mole fraction
  • y is the DMAEMA feed mole fraction
  • p the concentration of the APBA solution added (in mg/mL)
  • q concentration of EDC solution added (in mg/mL).
  • a Lexel 95 ion laser operating at a wavelength of 514nm and a power of 10OmW was used as the light source.
  • Correlation data was analyzed using a BI- 9000AT digital autocorrelator, version 6.1 (Brookhaven Instruments Corporation) and the CONTIN statistical method was used to calculate the particle size distributions. All microgels discussed in this study were monodisperse. Samples were typically prepared in thoroughly cleaned vials by suspending a small quantity of lyophilized microgel in (1) 0.005M pH9 carbonate buffer or (2) 0.005M pH7.4 phosphate buffer such that the scattering intensity was between 100 and 250 kilocounts per second. Glucose was added to selected samples in physiologically-relevant concentrations.
  • Physiological blood glucose levels of interest are within the range of 0-3 g/L, with 0.9-1.30 g/L (5.0-7.2 mM) being the "normal" blood glucose concentrations recommended for diabetics. Hypoglycemia occurs when blood glucose levels dip below 4mM (0.7g/L) while hyperglycemia occurs when blood glucose levels rise above 1 ImM (2.0g/L). A practical drug delivery vehicle or in vivo glucose sensor must show a significant response within this range.
  • Electrophoretic Mobility Electrophoretic mobility values were measured using a ZetaPlus analyzer (Brookhaven Instruments Corporation) operating in phase analysis light scattering mode. Samples were prepared as described earlier for dynamic light scattering. A total of 10 runs (each comprised of 15 cycles) were conducted; the experimental uncertainties represent the standard error of the mean of the replicate runs.
  • Insulin Uptake Insulin Uptake'. Insulin solutions were made by dissolving 2mg/mL insulin in a 0.02M hydrochloric acid solution and adjusting the pH to 4 by back-titrating the solution with sodium hydroxide. Insulin uptake was tested in both pH 4 (citric acid) and pH 7.4 (phosphate) buffers with ionic strength 1OmM. The pH-corrected insulin solutions were diluted in the appropriate buffer to a concentration of lmg/mL and uptake solutions were prepared by mixing 4.5mL of the resulting solution with 0.5mL of a 2mg/mL microgel suspension. This procedure results in insulin concentrations of 0.9mg/mL and microgel concentrations of O.lmg/mL in the uptake testing conditions.
  • the insulin-microgel solution was mixed on a rotary mixer overnight and then centrifuged to separate the microgel-insulin complexes from the soluble insulin.
  • the bicinchoninic acid (BCA) assay was used to determine the concentration of residual insulin present in the centrifuge supernatant. 2ml of the BCA reagent (containing bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0. IM NaOH, final pH 11.25) and 0.04mL of a 4% copper (II) sulphate pentahydrate solution were added to 0.1ml of the supernatant. The solution was immediately vortexed for 5 seconds and placed on the rotary mixer for 2 hours at 25 0 C.
  • BCA bicinchoninic acid
  • the absorbance of the resulting solution was measured at 562nm using a UV spectrophotometer (Beckman DU800). Reference solutions were also prepared by substituting O.lmL of buffer in place of the centrifuge supernatant, accounting for the light green coloration of the BCA assay solution itself. The absorbance readings from the supernatant solutions were subtracted from this blank reference to give a net absorbance reading. The readings were converted to concentrations using a calibration curve developed by applying the BCA assay strategy to insulin solutions of known concentration over the linear range of the assay (0.2-1. Omg protein/mL). Three replicate measurements are performed for each microgel to confirm the accuracy of the results.
  • a series of microgels were loaded with insulin using the uptake procedure described above in a pH 4 citric acid buffer solution. After centrifugation, the supernatant is assayed for insulin uptake while the pelletized insulin-microgel complex is resuspended in pH 7.4 phosphate buffer (1OmM). To simulate physiological conditions, 0.15M NaCl solution was added to 2 test tubes containing the pelletized gel while 0.15M NaCl and 2mg/ml glucose was added to a second pair of tubes. The solutions were redispersed and placed hi a shaker bath overnight at 37°C to simulate physiological release.
  • microgel phase was then removed via filtration through a 0.2 ⁇ m syringe filter pre-incubated with bovine serum albumin to block insulin adhesion to the filter.
  • This method gives uptake predictions within the experimental error of the uptake values measured using ultracentrifugation as the microgel separation technique.
  • the filtrate solution is analyzed for insulin content using the BCA assay as outlined in section 9.2.2.
  • E. coli. cells are cultured by adding ImL of the JM-109 strain into 5OmL of LB broth (1% tryptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0) in the presence of O.lmg/mL ampicillin. The cells were incubated under shaking (200RPM, 37°C) until the optical density reached ⁇ 0.6mg/mL. At this point, 0.5mg of isopropyl thiogalactoside was added to the suspension and incubation was continued for an additional two hours. Cells were harvested and washed via four successive centrifugation cycles into 1OmM pH 7.4 phosphate buffer and stored in a lOmg/mL suspension at 4 0 C until use. AU flocculation experiments were completed within four days of cell culturing to ensure cell viability during the experiment.
  • Yeast cells were also cultured by inoculating ImL of the Pichia pastoris in 5OmL of YEPD medium (1% yeast extract, 2% peptone, 2% dextrose). Cells were incubated by shaking at 200RPM overnight at 30°C. An aliquot of 50 ⁇ L of the resulting seed cells is then transferred to 5OmL of YEPD medium and incubated in a shake flask for 24 hours at 30°C. Cells were harvested, cleaned, and stored using the procedure previously described for E. coli.
  • Type II Saccharomyces cerevisiae yeast cells were purchased from Sigma- Aldrich and suspended at lOmg/mL in a 1OmM pH 7.4 buffer prior to experiments.
  • Cell Flocculation Experiments Cell flocculation experiments were performed using a Malvern Mastersizer 2000. In each experiment, 6OmL of buffer (pH 4 citrate, pH 9 carbonate or pH 10 carbonate, all with ionic strength 5OmM) was added to the instrument reservoir and pumped through the optical cell to align the instrument, hi a typical experiment, 0.5mL of the lOmg/mL cell suspension was added to the reservoir and pumped through the optical cell at a rate of ⁇ 4mL/s to measure the size distribution of the cell suspension prior to polymer or carbohydrate treatment. Beam obscurations for each cell type tested ranged between 5-15% at this concentration.
  • buffer pH 4 citrate, pH 9 carbonate or pH 10 carbonate, all with ionic strength 5OmM
  • Glucose, cellobiose, or sucrose was then added to the desired concentration via addition of a 300mg/mL sugar stock solution prepared in water. After a five minute wait time, the size distribution of the cells was again measured. A background salt concentration of 45OmM was also added in some experiments to test the effect of ionic strength on the selective flocculation process. Finally, 0.5mL of a lmg/mL microgel suspension was added to induce flocculation and the size distributions were measured at 10-30 minute intervals. This results in a mass ratio of 10: 1 dry cells:dry microgel for a typical flocculation experiment. The suspension was mixed gently under magnetic stirring to ensure floes circulatation throughout the measurement. Each size distribution measurement was repeated three times, with the reported results representing the average of the distributions generated.
  • samples were withdrawn at pre-defined intervals during the flocculation process for turbidity measurements using an HF Scientific DRT-15CE turbidmeter. Microgels scatter some light but in all cases account for ⁇ 2% of the total laser obscuration and turbidity values. Furthermore, while the addition of glucose or sucrose
  • SUBSTITUTE SHEET (RHi F ?6) does provide a nutrient feedstock for cell growth and proliferation, the pH conditions used for the flocculation testing are typically not conducive to cell growth and the Mastersizer size profiles of both the yeast and E. coli. cells remain constant over at least a three-hour period in the presence of sugar. As a result, changes in turbidity and/or light obscuration can be related to the concentration of cells remaining in suspension.
  • VAA-NIPAM has both a highly surface-localized radial -COOH distribution and a well- isolated chain -COOH distribution; consequently, the conjugation reactions can proceed relatively unhindered by steric barriers, allowing for an extremely high conjugation efficiency to be achieved (close to 100% conjugation yield). This yield is significantly higher than those previously reported for microgels systems and illustrates the advantage of engineering the microgel morphology to maximize functional group accessibility in grafting applications.
  • the degree of conjugation achieved with AA-NIPAM is very similar to that achieved with FA-NIPAM. This suggests that the negative effect of the significantly lower degree of functional group surface localization in AA-NIPAM (as shown by the electrophoretic mobility measurements in Table 5) is offset by the much higher average separation of the -COOH groups within the polymer chains (AA is a monoacidic monomer).
  • AA is a monoacidic monomer.
  • much larger deswelling is observed upon conjugation with AA-NIPAM since the remaining, unconjugated -COOH groups are much less localized than in FA-NIPAM and the local charge-charge repulsion contribution to the gel swelling becomes minimal.
  • MAA-NIPAM has a relatively blocky chain distribution and a core-localized radial distribution of -COOH groups.
  • MAA-NIPAM exhibited the lowest degree of conjugation, no detectable change in the electrophoretic mobility before and after the conjugation, and a minimal deswelling upon conjugation since the few carboxylic acid groups which do react with APBA are located in the more highly crosslinked core of the microgel in which swelling/deswelling is elastically more restricted.
  • the covalent conjugation of APBA to the microgel as well as the efficiency of the microgel purification protocol used were both confirmed by using a control sample in which the APBA solution and microgel suspension were mixed but no EDC was added. As shown in Table 5, no APBA was detectable in the microgel by NMR after purification and no significant change was observed in either the particle size or mobility data before and after conjugation.
  • Figure 1 presents the observed changes in particle size (Fig. Ia) and light scattering intensity (Fig. Ib) of the four different -COOH-containing microgel/APBA conjugates.
  • the responsiveness of the microgels can be regulated by changing both the glucose concentration as well as the -COOH content of the microgel.
  • the microgel swells more and scatters less light in l.Omg/mL glucose solutions compared to 0.5mg/mL glucose solutions (although the volume ratio error bars do in some cases overlap).
  • the volume ratio error bars do in some cases overlap.
  • systematic and concentration-dependent responses can be achieved to changes in the external glucose concentration.
  • a VAA-NIPAM microgel with 8mol% VAA was synthesized and subjected to the same conjugation conditions.
  • the volume phase transition can be used to (a) enhance the responsiveness of the conjugate microgels to glucose and (b) tune the APBA-microgel "sensor" to respond differently at different target glucose concentrations.
  • Thermal sensitivity also allows one to tune the glucose concentration range over which significant swelling and/or light scattering intensity responses are observed.
  • Figure 3 shows how the temperature can strongly affect not only the magnitude but also the trajectory of the scattering intensity and/or volume dependence on glucose concentration.
  • Glucose concentrations are chosen to represent physiologically-relevant situations; 0.4g/L is the hypoglycemic limit, 0.7-1.3g/L is the normal glucose concentration in blood, and 2g/L is the hyperglycemic limit.
  • the VPTT of the microgel in the absence of glucose lies below the test temperature while the VPTT at all glucose concentrations lies above the test temperature; consequently, significant swelling is observed when a small amount of glucose is added but higher glucose concentrations have a minimal impact on gel swelling since only the Donnan equilibrium mechanism induces swelling (ie. minimal thermal swelling occurs).
  • the microgel VPTT at glucose concentrations ⁇ 1.3g/L also lies below the test temperature; consequently, a large, thermally-assisted swelling is observed as the glucose concentration increases from 1.3g/L to 2g/L (interestingly, the range over which insulin release would be particularly desirable to avoid hyperglycemia).
  • any functionalized microgel the swelling response to a specific stimulus can be maximized by increasing the concentration of the environmentally-responsive functional groups (increasing the specific driving force for swelling) and/or decreasing the crosslink density (reducing the elastic resistance to swelling).
  • Table 6 shows the volumetric swelling and light scattering responses for a 22mol% acrylic acid functionalized microgel containing lmol% crosslinker and different APBA/PBA contents.
  • the AA-functionalized microgel used as the grafting platform in Figure 1 contains 6.5mol% acrylic acid and 4.5mol% crosslinker. The higher the APBA or EDC concentrations used in the synthesis procedure, the more PBA groups are grafted to the microgel.
  • the temperature and glucose concentration range over which the glucose-induced phase transition can be thermally enhanced can also be controlled exploiting the thermosensitive swelling transition of the microgels.
  • Table 7 shows how the density of PBA grafts to a specific platform microgel can regulate the swelling response.
  • APBA-AA-Il Al E5 1.26 + 0.15 0.81 ⁇ 0.01 1.37 ⁇ 0.09 0.82 ⁇ 0.01 APBA-AA-I l Al ElO 1.60 + 0.12 0.69 + 0.01 4.25 + 0.30 0.84 + 0.01 APBA-AA-I l A5 ElO 3.11 + 0.27 0.65 + 0.01 3.72 ⁇ 0.21 ⁇ 0.90 + 0.02 APBA-AA-I l A5 E20 7.65 ⁇ 0.33 0.56 + 0.01 1.26 + 0.13 1.05 + 0.03
  • the VPTT of the unmodified 1 lmol% AA-NIPAM microgel platform is ⁇ 60°C, well above both test temperatures. As more APBA or EDC is added to the conjugation mixture, more APBA is grafted to the microgel, making the microgel more hydrophobic and reducing the VPTT. At 25°C, the VPTT of each of the APBA-modified microgels lies above the test temperature, resulting in a systematic increase in gel swelling and decrease in the light scattering intensity in the presence of lmg/mL glucose. A similar trend exists at 37°C for the two microgels with the lowest APBA content.
  • the PBA contents of both the A5 ElO and A5 E20 microgels are sufficiently high such that the VPTT of these microgels lie close to (A5 ElO) or below (A5 E20) the 37 0 C test temperature.
  • the glucose response is restricted in the A5 ElO microgel and effectively "shut off' in the A5 E20 microgel by the thermal phase transition.
  • the temperatures at which both thermal amplification and suppression of the glucose- triggered phase transition occurs can be tuned based on the PBA content of the conjugate.
  • Similar control over the VPTT can be exercised by adjusting the number of non- conjugated carboxylic acid functional groups in the microgel.
  • conjugates can be prepared which contain the same number of PBA groups (and thus show the same ionization-based glucose response) but exhibit thermally-enhanced glucose swelling at higher temperatures.
  • Figure 4 shows how the functional monomer used to prepare the base microgel can also be used to tune the nature of the glucose-induced swelling.
  • APBA-AA-6.5 shows a clear VPTT shift, facilitating a thermally- enhanced five-fold swelling upon exposure to lmg/mL glucose at 27.5°C, and maintains at least some glucose swelling activity at temperatures as high as 35 0 C.
  • APBA-FA-6.5 exhibits no significant VPTT shift, swells no more than two-fold by volume in the presence of lmg/mL glucose at any temperature, and loses all glucose activity at T > 30 0 C.
  • tin ' s disclosure can tune the VPTT of the resulting microgel to either amplify or suppress the glucose-induced phase transition within specific glucose concentration and temperature ranges by controlling the content and distribution of graft sites in the platform microgel (via the polymerization recipe used) and the degree of APBA conjugation in the graft microgel (via the EDC/APBA concentration), hi addition, several different temperature switches can be prepared from the same platform microgel using different conjugation conditions in the graft-to approach described, facilitating the rapid synthesis of multi-temperature responsive microgel mixtures if desired.
  • microgels described to this point show glucose sensitivity only within ⁇ 1 unit of the pK a value of the phenylboronic acid functional entity ( ⁇ 8.9).
  • activity is required at physiological pH (7.4), temperature (37°C), and salt concentrations (tonicity equivalent to a 0.15MNaCl solution). It is believed that no previous glucose-sensing gels have been demonstrated to operate under such conditions.
  • salt concentrations such as sodium bicarbonate
  • Grafting PBA to amphoteric microgels has the same general effect as described earlier for anionic microgels: as the EDC and, to a lesser extent, APBA concentrations are increased, the graft yield of PBA groups increases and more free -COOH groups are consumed.
  • the surface charge on the microgel may switch from negative to positive at pH values close to the pK a of the PBA functional groups, hi this case, the ionization of PBA functional groups upon exposure of the microgels to glucose actually decreases the net charge density within the microgels (effectively "neutralizing" some of the cationic charges), causing the microgels to instead contract in the presence of glucose.
  • This behaviour which is believed to have never been previously reported, is illustrated in Figure 5 for the APBA(+)-17-4 microgel at physiological pH.
  • SUBSTiTTUTE SHEET (RULE 26) At low APBA/EDC concentrations (APBA(+)-17-4 Al ElO), few PBA residues are grafted to the microgel and the net surface charge of the gel in the absence of glucose (as estimated by the electrophoretic mobility data) remains negative; consequently, the gel swells, but only minimally, upon exposure to glucose. As the APBA/EDC concentrations are increased, more -COOH groups are consumed and, since only a fraction of the PBA residues are ionized at pH 7.4, the surface charge of the gel in the absence of glucose becomes positive.
  • APBA(+)-27-7 has significantly more cationic functional groups as well as a lower -COOH/-N(CH 3 ) 2 ratio when compared to the other two microgels.
  • the average pK * of the PBA groups in this microgel is significantly lower and more PBA functional groups are ionized at pH 7.4.
  • the net cationic charge density observed at Omg/mL glucose is significantly lower and a much higher degree of swelling is observed in the presence of glucose at pH 7.4.
  • enough PBA groups are ionized in this case such that the net charge density switches from positive to negative upon glucose exposure, resulting in positive microgel swelling and a two-fold increase in the microgel volume in the presence of lmg/mL glucose.
  • the observed pH-sensitivity ofthe glucose response can be applied to achieve super- responsive glucose microgel swelling.
  • a fixed concentration of lmg/mL glucose four-fold (APBA(+)-17-4 A50 E50) to ten-fold (APBA(+)-22-5 A5 E50) changes in volume are achieved simply by changing the pH ofthe microgel suspension from 7.4 to 9.0.
  • the glucose concentration can be used to enhance pH swelling in PBA-microgels.
  • Both the absolute and relative magnitudes of the glucose swelling (or deswelling) responses in amphoteric PBA-microgels can therefore be tuned by controlling the ratio and total number of cationic and anionic functional monomers used to prepare the platform microgels and the amount of PBA grafted to the microgels.
  • Figure 7 shows the hydrodynamic volume and light scattering intensity responses of an anionically-initiated microgel at pH 7.4 and pH 9.
  • the net surface charge on the microgel at pH 7.4 becomes less negative until eventually switching to positive at high degrees of conjugation, at which point the microgel shrinks instead of swells upon glucose exposure.
  • This negative- to-positive surface charge transition occurs at higher degrees of APBA conjugation than with the cationically-initiated microgels since the residual initiator fragments bound to the microgel are anionic instead of cationic and more -COOH groups must be consumed to allow the cationic charges to control the surface charge.
  • microgels which swell in response to glucose since more PBA can be conjugated without inverting the surface charge; conversely, cationically-initiated microgels are better for synthesizing microgels which shrink upon glucose addition since high PBA graft contents will yield more highly charged (and thus more highly responsive) microgels.
  • Figure 7 also illustrates the dramatic increase in swelling response which can be achieved by lowering the degree of crosslinking in the microgel.
  • the crosslinker feed used to prepare APBA(-)-22-5-2 is approximately 60% lower than that used in the preparation of the cationically-initiated microgels.
  • Even at the intermediate degree of conjugation the A5 E20 microgel, a three-fold volume increase and an 85% decrease in light scattering intensity is observed upon exposure to lmg/mL glucose. This is a significantly larger response than can be achieved with the cationic microgels even at the highest degree of conjugation tested (see Figure 6).
  • Each of these three microgels exhibits volumetric swelling (between 30-60% increases in volume), decreases in light scattering intensity, and more negative surface charge densities upon exposure to glucose under physiological pH and temperature conditions, exactly as required for the design of a self-regulating insulin release system.
  • Table 9 shows the behaviour of APBA(+)-22-5 A5 E50 microgel at ambient temperature. Table 9. Diameter, volume, and light scattering intensity response of the APBA(+)-22-5 A5 E50 microgel at various salt and glucose concentrations (pH 7.4, 25 0 C). The volume ratio and relative light scattering intensity are normalized based on the Omg/mL glucose and 0.15M salt concentration result.
  • the PBA-microgel conjugate behaves as a typical amphoteric polymer in that only a rninimal decrease is observed in the microgel diameter upon the addition of 0.15M NaCl. Indeed, the amphoteric microgels exhibit more narrowly dispersed particle sizes and enhanced colloidal stability in the presence of higher salt concentrations compared to the low ionic strength buffer.
  • the addition of glucose induces a significant, systematic swelling response, with a -40% volume increase occurring at lmg/mL glucose and a 110% volume increase occurring when 5mg/mL glucose is added. Consequently, this microgel system could be used "as is” in a laboratory environment to detect glucose concentrations in physiological fluids at ambient temperature, exploiting either or both of the particle size and light scattering responses to glucose.
  • Anionic-initiated microgels exhibit the same glucose-sensitive swelling responses at high ionic strengths while providing improved colloidal stability at physiological temperature to achieve "true” physiological swelling activity, as shown in Table 10.
  • APBA(-)-17-5-2 A5 E50 37 1.36 + 0.02 0.84 ⁇ 0.01 25 1.60 ⁇ 0.05 0.81 ⁇ 0.01 APBA(-)-17-5-2 A5 E20 37 1.76 + 0.07 0.88 ⁇ 0.02
  • Table 10 contract by ⁇ 10% upon the addition of 0.15M NaCl but remain colloidally stable under physiological conditions.
  • the anionic initiator system used to prepare these microgels is more effective at both stabilizing the microgel under the high ionic strength conditions and shifting the VPTT of the microgel above physiological temperature, preventing the aggregation observed with the cationic-initiated microgels at 37°C.
  • both microgels swell more in response to lmg/mL glucose at 37 0 C than at 25°C and the A5 E20 microgel swells more in response to glucose despite containing fewer PBA groups. Both of these observations are indicative of thermal amplification of the glucose-driven phase transition.
  • Macroscopic microgel deswelling is observed at ⁇ 35°C for the A5 E50 microgel and ⁇ 36°C for the A5 E20 microgel, both just below the test temperature of 37°C.
  • the VPTT shift observed upon glucose binding can be still applied at high ionic strengths to drive optimized changes in microgel diameter as the glucose concentration increases under high ionic strength conditions.
  • amphoteric microgel swelling can be both amplified and suppressed by the thermal volume phase transition.
  • Table 11 shows the volumetric swelling and light scattering intensity responses of amphoteric PBA-microgels containing increasing PBA contents.
  • Glucose-responsive swelling increases with the PBA content of the microgel until the 37°C test temperature is higher than the microgel VPTT both in the presence and absence of glucose.
  • glucose swelling in the A2 E20 microgel is driven primarily by ionic interactions
  • glucose swelling in the A2 E50 microgel is enhanced by the thermal phase transition
  • glucose swelling in the A2 ElOO microgel is suppressed by the thermal phase transition.
  • this response directly parallels the anionic microgel responses at low salt described hereinbefore. This observed thermal phase transition enhancement of the glucose-driven phase transition is particularly useful in the high ionic strength physiological environment.
  • AMPH(-)-28-8-2 A2 E20 -2.53 ⁇ 0.03 1.27 + 0.03 1.38 + 0.02 AMPH(-)-28-8-2 A2 E50 -0.60 ⁇ 0.04 0.98 + 0.02 1.01 + 0.01 AMPH(-)-28-8-2 A2 E100 +0.59 + 0.06 0.91 + 0.01 0.83 + 0.01
  • An amphoteric microgel with a net negative charge in the absence of glucose (the A2 E20 microgel) swells systematically when exposed to 1 mg/mL glucose while a microgel with a net positive charge prior to glucose addition (the A2 ElOO microgel) deswells systematically with the glucose concentration.
  • PBA-functionalized microgels can induce the flocculation of both E. coll and P. pastoris cells under a range of solution conditions.
  • the particular biotechnology usefulness of this strategy lies m the use of glucose to regulate the flocculation of cells via two possible mechanisms.
  • amphoteric PBA-microgels are used as the flocculant, cell flocculation can be induced electrostatically via inversion of the microgel surface charge as the glucose concentration increases.
  • Figure 8 shows the particle size distributions of E. coli. suspensions when two different PBA-functionalized amphoteric microgels are added in the presence of different glucose concentrations.
  • Table 13 gives the corresponding electrophoretic mobilities for each of the flocculating microgels and the E. coli. bacteria as a function of glucose concentration. Flocculation is indicated by the appearance of large particles in the particle size distributions.
  • E. coli. flocculation is observed at low glucose concentrations while no significant flocculation occurs at high glucose concentrations.
  • the surface charge on the microgel switches from cationic at low glucose levels to anionic at higher glucose levels as the PBA equilibrium shifts and more anionic PBA sites are generated. Since E. coli. cells are highly anionic at the physiological test pH, the microgels and cells would electrosterically repel each other at high glucose concentrations and electrostatically attract each other at low glucose concentrations. This charge inversion mechanism permits on-off switching of cell flocculation as a function of glucose concentration. Without intending to be bound by theory, the probable mechanism of glucose-controlled cell flocculation of E. coli. is illustrated in Scheme 1.
  • the cells flocculate in the presence of lOmg/mL glucose and 20mg/mL glucose is required to block flocculation.
  • the PBA content of the microgel can be used to control the interactions between the microgels and the cells in the absence of glucose.
  • Figure 9 shows the particle size distributions of E. coli. cells in the presence of microgels containing varying PBA contents.
  • Both the A5 E50 and A5 ElOO conjugated microgels are cationic in the absence of added glucose (Table 13) such that E. coli. flocculation is induced via the electrostatic mechanism of Scheme 1.
  • the A5 E20 microgel carries a net anionic charge in the absence of glucose and, correspondingly, does not induce cell flocculation.
  • the particle size profile given in Figure 9 shows two distinct peaks corresponding to both the unflocculated E. coli. cells (centred at ⁇ l ⁇ m) and the free microgel particles (centred at ⁇ 0.15 ⁇ m). This suggests that like surface charges on the microgel and the cell not only block cell flocculation but also switch off all significant interactions between the microgels and the cell surface.
  • This glucose-responsive charge inversion flocculation strategy could be applied for the selective harvesting of cells at specific phases in their growth cycles.
  • the glucose concentration added during cell culturing and/or the PBA content of the flocculating amphoteric microgel can be tuned to flocculate the growing cells at a selected interval in their growth cycle.
  • P. pastoris cells can also be flocculated by a specific PBA-cell surface carbohydrate binding mechanism.
  • Figure 10 reports the particle size distributions for the P. pastoris suspensions before and after microgel addition in the presence of glucose while Table 14 shows the corresponding relative turbidities of the cell suspensions and Table 15 reports the relevant electrophoretic mobilities of both the cells and the microgels under the flocculation conditions.
  • Table 15 indicates that both the microgels and the cells carry significant anionic charges at all tested glucose concentrations at pH 9, the pH value used for the P. pastoris flocculation experiments. Consequently, based on the lack of interaction observed in Figure 9 between microgels and cells carrying like charges, neither cell flocculation nor microgel-cell interactions would be anticipated under these conditions. However, floes become visible in the particle size distributions ( Figure 10) and tlhe relative turbidities the cell suspensions significantly decrease (Table 14) as PBA-microgels are added to P. pastoris suspensions. These observations suggest that the PBA-functionalized microgels can mediate P. pastoris flocculation in the absence of glucose.
  • the difference in the flocculation behaviors observed in the A5 E20 and A5 ElOO microgels in Figure 10 can also be understood based on the competitive binding mechanism.
  • the A5 E20 microgel At lower PBA loadings (the A5 E20 microgel), less glucose is required to effectively block all of the available PBA sites on the microgel.
  • cell flocculation is significantly limited at 2mg/mL glucose and entirely turned off at lOmg/mL glucose.
  • more PBA groups are present and more glucose is required to competitively inhibit PBA-cell surface carbohydrate interactions.
  • the degree of cell flocculation observed upon microgel addition can also be tuned according to the overall PBA graft content in the microgel, as shown in Figure 11.
  • Figure 12 shows how the APBA-22-0 A5 El 00 microgel can flocculate P. pastoris cells but cannot flocculate E. coll cells in a pH 9 salt solution.
  • E. coll is comprised primarily of peptidoglycan, the two major components of which (N- acetylglucosamine and N-acetylmuramic acid) do not contain any cis- ⁇ o ⁇ functional groups.
  • a variety of lipopolysaccharides (some of which may contain cis-diol groups) also protrude from this surface into the solution.
  • yeast cells like P.
  • the cell wall is comprised primarily (>80%) of glucans and mannans, both of which contain c ⁇ -diol groups. Furthermore, the glycocalyx protruding from the cell surface is rich in glycolipids and glycoproteins which contain cis-diol stereochemistry.
  • sucrose does significantly slow the flocculation rate and reduces the size of the resulting floes compared to the case in which no carbohydrate inhibitor is added.
  • the higher the PBA-carbohydrate binding constant the more successfully the carbohydrate competes with cell surface carbohydrates to inhibit flocculation and the slower the flocculation rate.
  • the PBA groups are ionized in their tetrahedral, glucose-binding form and both the microgel and the yeast carry large anionic charges. Consequently, electrostatic repulsion keeps the microgel and yeast apart to minimize non-specific adhesion while glucose can competitively inhibit the specific PBA-cell surface carbohydrate interactions driving the yeast flocculation. Indeed, while ⁇ 1% flocculation is observed at both pH 9 and pH 10 in the presence of 20mg/mL glucose, nearly 100% flocculation efficiency is observed at both pH 9 and pH 10 if glucose is not added to block the specific PBA-cell binding. However, at pH 4, the surface charges on both the yeast and the microgel are significantly lower and some non-specific flocculation is observed.
  • the selectivity of the PBA-cell interactions can effectively be tuned by the external salt concentration, the pH of the suspension, and the type of inhibiting carbohydrate added.
  • This multi-variable control over cell flocculation may be useful for controlling the flocculation rate, the critical glucose concentration for turning the flocculation "on” and “off', and the size the resulting cell aggregates.
  • the concept of selective PBA-mediated flocculation may be extended to a number of other applications.
  • Cells grown under different medium conditions may be differentiated since the nature of the growth medium can influence the carbohydrate composition at the surface of cells.
  • Biological signals may be mediated via specific cell-microgel interactions given the importance of cell surface carbohydrates in regulating cell signaling events.
  • Different types of carbohydrates can also be targeted by changing the chemical structure of the PBA binding group used. As an example, using a cyclic boronic acid increases the cone angle in the boronic acid residue to enhance the selectivity of boronic acid binding to glycoside carbohydrates over other sugars.
  • mammalian cells expressing specific carbohydrate markers may be targeted using boronic acid-functionalized microgels, a useful technology for both cell recovery and cell signaling.
  • boronic acid-functionalized microgels a useful technology for both cell recovery and cell signaling.
  • a variety of fluorescent PBA residues have also been designed which would facilitate the selective imaging of specific cell types in complex mixtures.
  • SUBSTiTUTC SHEET (RULE 26) Targeted drug delivery should also be possible by tuning the PBA-microgels to bind to cells displaying specific carbohydrate markers.
  • the PBA functionality of the microgel can anchor the delivery vehicle to a targeted cell surface while the gel phase can control the release rate of a specific drug at the target site.
  • PBA binding to a cis-diol surface would also shift the PBA equilibrium to generate more anionic PBA sites, enabling control over drug release via both the diffusion and charge-controlled mechanisms described in Scheme 1.
  • a typical marker of cancer cells is the over-expression of sialic acid, a cis-diol containing carbohydrate with a high affinity binding with PBA.
  • the high sialic acid content in mucous membranes may be applied to use PBA-microgels as drug delivery vehicles for the oral or nasal delivery of pharmaceutical vehicles.
  • PBA-microgels as glucose sensors is contemplated, flowing from the systematic diameter and light scattering changes observed as the glucose concentration is increased in both low salt ( Figure 3) and high salt (Table 12) conditions, hi particular, thermally-enhanced glucose swelling observed within specific glucose concentration ranges in Figure 3 is beneficial to produce sensors with enhanced glucose responses over targeted glucose concentration ranges.
  • Figure 16 shows that linear diameter and light scattering intensity responses can be achieved with the microgel sensors over specific ranges of glucose concentration by controlling the PBA content of the microgel.
  • microgels could also be applied in the construction of a microgel sensor device comprised of a three-dimensional colloidal array of the highly monodisperse PBA-microgels constructed using well-defined strategies. Glucose concentration changes could then be tracked both by changes in the overall turbidity of the microgel film and the color of light refracted from the highly ordered microgel arrays as the inter-lattice spacing changes with the degree of swelling in the individual glucose- responsive microgels.
  • the observed adjustability of the glucose concentration response range based on the PBA content of the microgel further suggests the potential of tuning the glucose sensors for different glucose-containing environments.
  • the glucose concentration in the intracellular space is slightly lower than that of the arterial blood (normally ⁇ 3.9-4.8mM as opposed to 5.2-7.2mM) while the normal glucose concentration in tears is 5-10-fold lower than that in blood.
  • Both concentrations are directly related to the blood glucose level and could be used to indirectly measure blood glucose and/or (particularly in the intercellular region) regulate drug release.
  • the pH of 7.2-7.3 in these environments is still sufficiently high to generate glucose responses in the amphoteric microgels.
  • the PBA-microgels should be useful for both laboratory and in vivo sensing in a range of different environments.
  • the equilibrium will shift back toward the uncharged PBA species and the gel will deswell, reducing the diffusion rate of insulin out of the microgel and avoiding "overshoots" in blood glucose correction.
  • the charge inversion mechanism relies on the switching of the net charge density in the microgel upon glucose binding.
  • Insulin is a protein of mass 5.5kDa and isoelectric point of 5.4 and thus carries a negative charge at physiological pH. Therefore, microgels which carry a net cationic charge in the absence of glucose at physiological pH can bind insulin via electrostatic attraction.
  • the PBA equilibrium shifts to generate more anionic charges and switch the net charge on the microgel to anionic, driving insulin release via electrostatic repulsion.
  • Both mechanisms have a feedback mechanism (microgel deswelling in (a) or anionic charge density reduction in (b)) which reduce the rate of insulin release as the insulin acts and the glucose concentration in the environment is reduced.
  • the AMPH(-)-28-8 A5 E50 microgel was selected as a model system given its high PBA content (-70% of -COOH groups converted to PBA groups) and its high amine content (maximizing the potential for charge inversion and lowering thepK a of the maximum number of PBA groups).
  • the AMPH(-)-28-8 A2 E20 microgel which contains the same base functional group composition but a significant lower PBA content (-25% -COOH conversion) is also evaluated to test the effect of the PBA content on the insulin responses.
  • Table 16 shows the uptake and Table 17 shows the release of insulin achieved using this microgel as a function of both pH and glucose concentration and the corresponding surface charges on the microgel.
  • Microgel Electrophoretic Mobility Insulin Uptake (xl ⁇ ⁇ 8 mVVs) (mg insulin/mg microgel) pH 4, 10mM pH 7.4, IQmM pH 4, 10mM pH 7.4, 1OmM
  • AMPH(-)-28-8 A2 E20 -1.86 ⁇ 0.04 -1.71 ⁇ 0.02 6.18 ⁇ 0.05 0.26 + 0.12
  • Insulin release (% of insulin in each of the complexes described in Table-9.1) for amphoteric PBA-microgels of different PBA contents over 24 hours. Insulin loading was conducted using a pH 4, 1OmM citric acid buffer. Release experiments are performed under physiological conditions (pH 7.4, 37°C, 15OmM ionic strength) while the mobility values are measured in a 1OmM pH 7.4 phosphate buffer.
  • SUBSTITUTE SHEET Insulin uptake appears to be driven primarily by electrostatic interactions between the amphoteric microgels and the insulin protein.
  • insulin carries a net cationic charge while the microgels are anionic due to peptization by the divalent citric acid buffer salt. Consequently, extremely large insulin uptakes are observed in both microgels.
  • insulin carries a net anionic charge while the A2 E20 microgel is anionic and the A5 E50 microgel is cationic.
  • minimal insulin uptake is achieved with the A2 E20 microgel while a large insulin uptake is facilitated by the A5 E50 microgel.
  • the amount of insulin uptake is a function of the electrostatic interactions between, the amphoteric microgel and insulin at the pH value used for protein uptake.
  • the pH 4-loaded insulin-microgel complexes are redispersed under physiological conditions (pH 7.4, 15OmM ionic strength, 37°C)
  • insulin is released from both microgels.
  • the A2 E20 microgel shows no glucose-specific insulin release while the A5 E50 microgel (high PBA content) selectively releases more insulin in the presence of higher glucose levels.
  • APBA(-)-28-8 A2 E20 carries a net anionic charge at physiological pH regardless of the glucose concentration such that microgel-insulin repulsion occurs whether or not glucose is present and no significant difference is observed in the insulin release when glucose is added.
  • APBA(-)-28-8 A5 E50 carries a slight net cationic charge in the absence of glucose (pl ⁇ 7.8) and a slight net anionic charge in the presence of glucose (pl ⁇ 7.3). Therefore, anionic insulin is attracted to the microgel in the absence of glucose but repelled from the microgel when glucose is added, resulting in the increased observed insulin release at 2mg/mL glucose.
  • amphoteric PBA-microgels should be useful for facilitating glucose-responsive insulin release. To do so, one would need to improve the microgel pi shift observed upon glucose binding. This may be achieved by maximizing the PBA content of the microgel and, correspondingly, the functional group content of the base amphoteric microgel platform.
  • Diffusion-controlled release could also be promoted by controlling the crosslinker density and distribution such that the change in the average pore size as glucose is added significantly enhances the diffusion of the 5.5kDa insulin molecule out of the microgel matrix.
  • the crosslink density, particle size, and charge ratio in the microgel must be balanced to facilitate physiologically-appropriate release rates in vivo.
  • the formation of microaggregates may be desirable in this context to increase the overall size of the microgel and thus control insulin release over a longer period of time.
  • amphoteric microgel would be designed to mirror the charge density of a target molecule under the uptake conditions and parallel the charge density of a target molecule under the optimal release conditions.
  • electrostatic attractions between the microgel and the target are maximized under the uptake conditions while electrostatic repulsion between the microgel and the target are maximized under the release conditions.
  • This ability to tune the charge profile of the microgel to match that of a target molecule and/or target cell may be applied to extend amphoteric microgels as responsive binding agents for other proteins, pathogens or biomolecules with complex charge profiles.
  • Phenylbdronic acid-modified poly(N-isopropylacrylamide)-based microgels can be designed to exhibit reversible swelling responses to changes in the glucose concentration.
  • the underlying functional group distribution controls the relative changes in the turbidity and particle volume as glucose is added while the total number of PBA groups present in the microgel controls the absolute magnitude of the responses.
  • the thermal phase transition can be applied to both amplify and suppress ionization- driven glucose swelling. Both the temperature and glucose concentration ranges over which glucose phase transition amplification and suppression are achieved can be tuned by changing the underlying composition of the microgel.
  • Amphoteric PBA-functionalized microgels can be designed which can either shrink or swell in response to increases in the glucose concentration according to the solution pH and the cationic/anionic charge ratio in the microgel network.
  • Amphoteric microgels are colloidally stable under physiological conditions and can exhibit up to two-fold increases in volume upon exposure to lmg/mL glucose at pH 7.4, 37°C, and 0.15M ionic strength. This is believed to be the first reported microgel system to achieve such physiological responses.
  • Glucose sensors can be designed based on PBA-functionalized microgels which exhibit linear particle size and scattering intensity responses over specific glucose concentration ranges. These linearly-responsive glucose concentration ranges are tunable according to the PBA graft content in the microgel.
  • Amphoteric microgels can be designed to bind large amounts of insulin (-3-6 times the mass of the dry microgel in the suspension) by matching the charge profiles of the insulin and the amphoteric gel network at the uptake pH.
  • Glucose-sensitive insulin release should be possible by inverting the microgel charge Upon glucose exposure in physiological conditions.
  • Glucose can be used to selectively inhibit the flocculation of biological cells by PBA- functionalized microgels by inverting the electrostatic charge on the microgel surface and/or the competitively binding to PBA sites in the microgel to block the specific interaction between PBA groups and cis-dio ⁇ carbohydrates expressed. on cell surfaces.
  • P. pastoris cells can be selectively flocculated in the presence of E. coli. cells by exploiting differences in the cell surface carbohydrate compositions.
  • the ionic strength, pH, and carbohydrate inhibitor selected for the flocculation can be applied to tune the aggregation rate and the floe size of PBA-microgel/cell aggregates.

Abstract

La présente invention concerne un microgel fonctionnalisé, caractérisé par la présence de groupes fonctionnels d’acide phénylboronique (PBA). L’invention concerne également un processus de fabrication du microgel, qui comprend l’étape consistant à greffer de l’acide aminophénylboronique (APBA) à un microgel carboxylé via le couplage de l’EDC. Le microgel de cette invention peut également être fabriqué par la polymérisation d’un monomère contenant de l’acide phénylboronique (PBA). Le microgel peut être adapté afin de se dégonfler en présence de glucose; de se gonfler en présence de glucose dans des conditions physiologiques humaines ; de maintenir une charge de surface nette négative à une valeur de pH physiologique; en cas d’exposition au to glucose, soit de se gonfler soit de se contracter en réaction au pH; ou de contenir des charges à la fois anionique et cationique dans des conditions physiologiques. L’invention décrit également des utilisations du microgel dans le cadre du traitement de l’eau, de la séparation biologique, des détecteurs de glucose, de l’autorégulation de dispositifs de libération d’insuline et des commutateurs de fluides.
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WO2011113989A1 (fr) 2010-03-18 2011-09-22 Universidad Del País Vasco Nanogels cationiques pour applications biotechnologiques
WO2011159161A2 (fr) 2010-06-18 2011-12-22 Universiteit Twente Polymères borés
US8569231B2 (en) 2009-03-20 2013-10-29 Smartcells, Inc. Soluble non-depot insulin conjugates and uses thereof
US8623345B2 (en) 2009-03-20 2014-01-07 Smartcells Terminally-functionalized conjugates and uses thereof
US8846103B2 (en) 2009-01-28 2014-09-30 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
US8906850B2 (en) 2009-01-28 2014-12-09 Smartcells, Inc. Crystalline insulin-conjugates
US8933207B2 (en) 2010-07-28 2015-01-13 Smartcells, Inc. Drug-ligand conjugates, synthesis thereof, and intermediates thereto
US8940690B2 (en) 2009-01-28 2015-01-27 National Institutes Of Health (Nih) Synthetic conjugates and uses thereof
US9050370B2 (en) 2009-01-28 2015-06-09 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9068013B2 (en) 2010-07-28 2015-06-30 Smart Cells, Inc. Recombinant lectins, binding-site modified lectins and uses thereof
US9074015B2 (en) 2010-07-28 2015-07-07 Smartcells, Inc. Recombinantly expressed insulin polypeptides and uses thereof
CN106243442A (zh) * 2016-07-28 2016-12-21 上海超高环保科技股份有限公司 对含苯物质具有高过滤性的组合物
EP3250255A4 (fr) * 2015-01-27 2018-10-10 The Texas A&M University System Membrane auto-nettoyante pour dispositifs médicaux
WO2018223114A1 (fr) * 2017-06-02 2018-12-06 North Carolina State University Compositions sensibles au glucose pour l'administration de médicament
WO2020093173A1 (fr) * 2018-11-08 2020-05-14 Xiao Yu Wu Composition et dispositif pour prévenir l'hypoglycémie et leur utilisation
CN113603826A (zh) * 2021-06-30 2021-11-05 浙江大学 一种丙烯酰基甘氨酰胺-苯硼酸基糖敏微针的制备方法
CN115746196A (zh) * 2022-11-15 2023-03-07 电子科技大学长三角研究院(湖州) 一种异丙基甲基丙烯酰胺-氟基苯硼酸共聚葡萄糖响应微凝胶的制备方法及其应用
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US8846103B2 (en) 2009-01-28 2014-09-30 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
US9463249B2 (en) 2009-01-28 2016-10-11 Smartcells, Inc. Crystalline insulin-conjugates
US8906850B2 (en) 2009-01-28 2014-12-09 Smartcells, Inc. Crystalline insulin-conjugates
US10398781B2 (en) 2009-01-28 2019-09-03 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US8940690B2 (en) 2009-01-28 2015-01-27 National Institutes Of Health (Nih) Synthetic conjugates and uses thereof
US9050370B2 (en) 2009-01-28 2015-06-09 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9579391B2 (en) 2009-01-28 2017-02-28 Smartcells, Inc. Conjugate based systems for controlled drug delivery
US9114176B2 (en) 2009-01-28 2015-08-25 Smartcells, Inc. Exogenously triggered controlled release materials and uses thereof
US8569231B2 (en) 2009-03-20 2013-10-29 Smartcells, Inc. Soluble non-depot insulin conjugates and uses thereof
US8623345B2 (en) 2009-03-20 2014-01-07 Smartcells Terminally-functionalized conjugates and uses thereof
WO2011113989A1 (fr) 2010-03-18 2011-09-22 Universidad Del País Vasco Nanogels cationiques pour applications biotechnologiques
WO2011159161A2 (fr) 2010-06-18 2011-12-22 Universiteit Twente Polymères borés
US9074015B2 (en) 2010-07-28 2015-07-07 Smartcells, Inc. Recombinantly expressed insulin polypeptides and uses thereof
US9068013B2 (en) 2010-07-28 2015-06-30 Smart Cells, Inc. Recombinant lectins, binding-site modified lectins and uses thereof
US8933207B2 (en) 2010-07-28 2015-01-13 Smartcells, Inc. Drug-ligand conjugates, synthesis thereof, and intermediates thereto
EP3250255A4 (fr) * 2015-01-27 2018-10-10 The Texas A&M University System Membrane auto-nettoyante pour dispositifs médicaux
US11701455B2 (en) 2015-01-27 2023-07-18 The Texas A&M University System Self-cleaning membrane for medical devices
CN106243442A (zh) * 2016-07-28 2016-12-21 上海超高环保科技股份有限公司 对含苯物质具有高过滤性的组合物
WO2018223114A1 (fr) * 2017-06-02 2018-12-06 North Carolina State University Compositions sensibles au glucose pour l'administration de médicament
US11096893B2 (en) 2017-06-02 2021-08-24 North Carolina State University Glucose sensitive compositions for drug delivery
WO2020093173A1 (fr) * 2018-11-08 2020-05-14 Xiao Yu Wu Composition et dispositif pour prévenir l'hypoglycémie et leur utilisation
CN113603826A (zh) * 2021-06-30 2021-11-05 浙江大学 一种丙烯酰基甘氨酰胺-苯硼酸基糖敏微针的制备方法
CN113603826B (zh) * 2021-06-30 2022-06-28 浙江大学 一种丙烯酰基甘氨酰胺-苯硼酸基糖敏微针的制备方法
WO2023122497A1 (fr) * 2021-12-20 2023-06-29 University Of Florida Research Foundation, Inc. Microgels, procédés de fabrication de microgels et procédés d'utilisation de microgels
CN115746196A (zh) * 2022-11-15 2023-03-07 电子科技大学长三角研究院(湖州) 一种异丙基甲基丙烯酰胺-氟基苯硼酸共聚葡萄糖响应微凝胶的制备方法及其应用

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