1. Thermostabilization of inactivated polio vaccine in PLGA-based microspheres for pulsatile release
Stephany Y. Tzeng, Rohiverth Guarecuco, Kevin J. McHugh, Sviatlana Rose, Evan M. Rosenberg, Yingying Zeng, Robert Langer, Ana Jaklenec

2. Inactivated polio vaccine (IPV):
much safer than OPV (no risk of reversion to a disease-causing virus)
must be injected more than once for efficacy
coverage for a second dose < first dose
poor thermostability
Administered in D-antigen conformation: over time at high temperature, it converts to the C-antigen conformation
Controlled release microspheres: single injection, thermostability
Trans position: reduce d antigen expression due to steric hindrance from c antigen

3. IPV Thermostability Studies
*IPVconc
Dilute IPVconc serotype 1,2, and 3 in distilled water.
Mix with a sterile solution of an excipient. (1:1 v/v)
Aliquot mixtures into sealed vial and stored at 37’C.
At time points, remove vial and analyzed by ELISA by normalizing the D-antigen measurement
Concentrate tIPV using centrifuge (filters: 100kDa mw cutoff, 14,000 rcf for 10 min)
Dilute retentate with 0.5mL sterile distilled water
Concentrate once more, so remove the small molecule components
Final concentration: 8 DU/mL, 1.7 DU/mL, and 6.8 DU/mL type 1, 2, and 3. (approximately tenfold lower concentration than the normal concentration)

4. IPV Thermostability Studies
A. No excipient. Rapidly lose D-antigen content (30%,86%,66% after 1month)
B. Serotype 1: least stable, high contribution to paralytic poliomyelitis
Various polyols w/ MSG MgCl2
Negative except Trehalose
Sugar shows better results with MSG
Best thermostability: after adding MgCl2 (USE ALL TOGETHER)
C. Sucrose, Trehalos, Maltodextrin is effective in combination with MSG and MgCl2
Recovery decrease with decreasing excipient conc.
Sucrose 2.5 fold, Trehalose 3.6 fold, Maltodextrin less than half
Gelatin has destabilizing effect on IPV
D. After 2 months of incubation, IPV D antigen is stable with high conc. of maltodextrin, MSG, and MgCl2 (formulation for further microsphere)

5. IPV Processing Thermostability Studies: w/oil emulsion
Mix 19µL of IPVconc with 10µL of aqueous solution of excipients (100:1 excipient:IPV mass ratio)
Add mixture to 25mg PLGA50:50 in 1mL dichloromethane
Emulsify via sonication on ice (20% amplitude for 30s)
Add 2mL of ELISA dilution buffer, vortex
DCM layer is evaporated after 3hr stirring
Analyze remaining aqueous phase by ELISA by normalizing the D- antigen measurement
SURVIVE DURING EMULSION: contact with organic solvents and physical mixing stresses

6. IPV Processing Thermostability Studies: w/oil emulsion
Sugar, amino acids, or MgCl2 salt: no significant effect
gelatin: significant positive effect
major stresses: the increase in interfacial tension
gelatin: amphiphilic, shield protein from damage
major stresses: the increase in interfacial tension between the oil and water phases, cause denaturation and promote aggregation

7. IPV Processing Thermostability Studies: vacuum-drying
10 microliters water or aqueous solution of excipients was added to 19 μL of IPVconc (100:1 excipient:IPV mass ratio)
Place in a vacuum for 1 h at RT (DRYING)
Redissolved in ELISA dilution buffer and analyze
Percent recovery was calculated by normalizing the D-antigen measurement

8. IPV Processing Thermostability Studies: vacuum-drying
without excipients: 1.0 %, 15 %, and 1.8%
Gelatin: steric hindrance
Carbohydrate: native conformation, mobility
Trehalose and maltodextrin: most protection on their own.
Addition of MSG and MgCl2: recovery rates increased for the other sugars as well
(MgCl2 changed the solubility of the proteins in the IPV capsid)

9. Emulsion microsphere formulations (w/o1/o2)
*first emulsion
the w phase(tIPV+excipient) was added to the o1(PLGA) phase,
the mixture was sonicated
For formulations containing Mg(OH)2, solid Mg(OH)2 was dispersed in the o1 phase
add w and sonicating again.
*second emulsion
o2(heavy mineral oil) was added to the w/o1 emulsion and vortexed
Pour w/o1/o2 into stirring heavy mineral oil.
stirred for 3 h at RT (DCM evaporation).
Particles were collected by centrifugation.
washed three times with hexane.
dried for 1 h under vacuum at RT.
The dry microspheres were either resuspended in buffer for use.

10. Release studies for unlabeled IPV-containing microsphere
IPV microspheres were resuspended in release buffer(PBS).
The microparticle suspension were incubated at 37 °C on a tube revolver.
At predetermined time points, tubes of particles were centrifuged.
The supernatant of each tube was stored at 4 °C for up to 1 week before analysis.
The particles in each tube were resuspended in release buffer, vortexed, and returned to 37 °C until the next time point.
D-antigen in the collected supernatants was measured via ELISA.

11. Release studies for unlabeled IPV-containing microsphere
Gelatin: nearly 100% of all release occurring in the first few days, gelatin improved IPV recovery after emulsification and drying but showed very little ability to stabilize IPV in an aqueous, 37 °C environment over time
F2, F3: mainly of serotype 2 and only very low levels of serotypes 1 and 3, Both sucrose and maltodextrin in combination with MSG and MgCl2 are superior for long-term IPV stability inside PLGA particles in an aqueous 37 °C buffer,
IPV virions very near the surface of the particles are released quickly in an initial burst, and there is a delay of release while water enters the microsphere matrix and begins to degrade the PLGA by bulk erosion.

12. IPV pH-sensitivity studies
AF680 labeled IPV encapsulated in PLGA50:50 microspheres with 10 μL of 20% sucrose, 17% MSG, and 17% MgCl2
IPV release was measured by fluorescence
Salt solutions of varying pH were prepared.
1× PBS was used for all solutions, with small amounts of 1 M HCl or 1 M NaOH.
tIPV was diluted 1:200 in solutions of pH 1, 4.5, 6, 7.4, 8, or 9.
Stored at either 4 °C or 37 °C.
After 7 days, the dilute IPV solutions were neutralized with either HCl or NaOH.
D-antigen recovery was measured via ELISA.

13. IPV pH-sensitivity studies
C pH can have an important effect on its stability
At low concentrations, as IPV would be after release into the buffer, type 1 IPV loses stability rapidly even at neutral (7.4) Ph
2 and 3 both tended to be more stable than type 1, with no statistically significant loss in D-antigen content after 7 days of incubation at pH 7.4
type 2 stability decreased upon incubation in an acidic medium, with only 31 ± 5% recovery after 7 days at pH 6
type 3 appeared to be slightly less sensitive, ), lost stability when kept in even slightly basic pH

14. Immune response to IPV in rodents
upon HYDRATION EPO cause ph increase, accelerate degradation by base-catalyzed hydrolysis, EPO protonate in acidity increase, EPO dissolved into aqueous phase and diffuse out, create channel)
Doping EPO: improve release of stable IPV
2nd burst span: large portion 40% (due to EPO accelerate degradation-near-complete IPV release in a week)
Can tune release kinetic easily by control ratio of EPO to PLGA (7.5% is short term 2nd release)

15. Immune response to IPV in rodents
1.4
1.1
1.2
within the 1–2 month timeframe that is currently recommended by the WHO.
0.5
0.8
0.6

16. Efficacy of IPV-containing microspheres in vivo
Rat IM
Microsphere: adjuvant
F8 Surpass 2 dose bolus
MS 1st : similar ab to single bolus, (same total IPV dose bur less release)
Single drop quickly
elicited high antibody titers within a few weeks and then maintained this high level for several weeks,
stronger antibody response compared with a single bolus.

17.
Super nice
EPO helps stable IPV release? (ELISA? Fluorescence?)
Mention FIG9 as FIG8
No clear signs of toxicity were observed after injection with microspheres. (in vivo)