Thermoresponsive Polymers for Regulating Vaccine Adjuvants

An adjuvant is a substance added to a vaccine to enhance immune response to the antigen
Adjuvanted vaccines are more likely to cause inflammatory side-effects such as fever, headache, and redness and swelling at the injection site(1)
Lipopolysaccharides (LPS) are glycolipids that can be found on the cell wall of gram-negative bacteria
LPS is a toll-like receptor 4 (TLR4) agonist and potent immunostimulant, however it is currently limited in its use in vaccines due to its reactogenicity, or ability to cause inflammatory side effects

NIPAM

Poly(N-isopropylacrylamide) (NIPAM) is a polymer that changes conformation from a coiled state to a globular state above its lower critical solution temperature (LCST) near human body temperature(2)
It has been copolymerized and examined as a drug delivery system(3)
It has also been used before to sequester LPS from an aqueous solution, though whether or not LPS’s incorporation into the polymer phase attenuates its immunogenicity remains untested(4)

As a Thermoresponsive Regulator

NIPAM’s thermoresponsive properties could be used to create a negative feedback system to reduce fever
The Mancini lab has previously created a NIPAM-adjuvant copolymer for this purpose(5)
This project examines if NIPAM can reduce the immunogenicity of LPS above its LCST by incorporating it into its polymer phase for the purpose of creating a thermoresponsive vaccine adjuvant system

In the upper portion of the image labeled "Below LCST," a representation of LPS labeled "LPS" and a representation of NIPAM labeled "NIPAM (C)" lay outside of a lipid bilayer with a representation of TLR4 attached. LPS is attached to TLR4 and is shown to produce a "Pro-Inflammatory Response." In the bottom half of the image, LPS is represented as encapsulated by a ball of NIPAM. This representation is labeled "NIPAM (G) + LPS." LPS is not shown interacting with TLR4 and producing a "Pro-inflammatory Response."

Figure 1. Representation of NIPAM theoretically enveloping LPS above its LCST

Below LCST, LPS and the coiled (C) form of NIPAM are in free solution together. LPS activates TLR4 on the cell surface, leading to a pro-inflammatory response. Above LCST, LPS is enveloped by globular (G) NIPAM, and being sequestered from solution, fails to activate TLR4 and the pro-inflammatory response

Research Questions

Can NIPAM sequester LPS above its LCST?
Can NIPAM attenuate LPS's ability to produce an immune response above its LCST?
What is the optimal concentration of NIPAM to exhibit a sharp LCST and attenuating effect?

Methods

Cell Culture

RAW-Blue cells cultured at 37℃, 5% CO2
Media was changed after 1 and 3 days and cells were passaged at 80% confluency

NIPAM Polymerization

NIPAM was polymerized via Reversible Addition-Fragmentation Chain-Transfer (RAFT) polymerization
Polymer molecular weight: 22,374 g/mol

Quanti-Blue Assay

Measures secreted embryonic alkaline phosphatase (SEAP), an indicator of TLR4 activation and immunogenic response
Cells incubated with varying concentrations of LPS, NIPAM, both in equal concentrations, or neither in 96-well plates for 48 hours at 37℃ (Eppendorf incubator ) or 40℃ (Lionheart microscope) (Figure 4, 5, and 6)
Both plates were incubated at 37℃ with uniform LPS concentrations in Eppendorf incubator and Lionheart microscope respectively (Figure 7)
Supernatant was transferred to new 96-well plates with quanti-blue reagent
Absorbance at 630 nm was read on a plate reader

The chemical structure of 2-(Ethylthiocarbonothioylthio) propanoic acid is labeled "A." The chemical structure of the NIPAM monomer is labeled "B." The chemical structure of the NIPAM polymer is labeled "C."

Figure 2. Polymerization of NIPAM

The chain transfer agent (CTA) 2-(Ethylthiocarbonothioylthio) propanoic acid (A) was reacted with the NIPAM monomer (B) and AIBN in DMF/DMSO at 65℃ to produce the polymer (C)

In the first box labeled "1," NIPAM and LPS are shown to be added to a 96-well plate with cells and that plate is put in either an incubator or an incubated microscope. In the next box over, labeled "2," the supernant from that plate is shown to be added to a new plate with "QB Reagent." In the next box over, labeled "3," a blue 96-well plate is shown to be read by a plate reader at 630 nm. In the bottom box, LPS is shown to bind to the dimer TLR4 on the cell surface. A pathway is then shown going to the nucleus of that cell, activating "NF-kB1," causing a transcription of the SEAP reporter gene to occur. A pathway is shown where mRNA moves out of the nucleus to the rough ER. From the rough ER, there is an arrow pointing to "SEAP" outside of the cell where it comes into contact with "Quanti-blue reagent (BCIP p-toluidine salt)."

Figure 3. Representation of quanti-blue assay procedure and cellular mechanism

LPS variably activates TLR4 pathway resulting in NF-κB activation, transcription of SEAP reporter gene, and SEAP synthesis and exocytosis (1). SEAP reacts with quanti-blue reagent to produce a fluorescent product (2)

Results

Results are shown in the figures below.

Graph shows absorbance plotted versus concentration in pg/mL for 35C, 37C, and 40C. All lines trend upward, except for 40C at 1 pg/mL. The y axis ranges from 0 to 1.2 and the x axis ranges from 1 to 100,000. 35C is generally higher than 37C and 37C is generally higher than 40C. There is one star above 35C at 1,000 and 10,000 pg/mL and two stars above 37C at 1,000 pg/mL. There is a star in between 35C and 40C at 100,000 pg/mL.

Figure 4. SEAP for cells incubated with LPS alone

(n = 3, *p<0.05, **p<0.01, ***p<0.001) SEAP increases with concentration of LPS as expected. Cells incubated at 35 ℃ show significantly higher SEAP than those incubated at 37℃ and 40℃ at 1,000 pg/mL and 10,000 pg/mL LPS

Graph shows absorbance plotted versus concentration in pg/mL for 35C, 37C, and 40C. The x axis ranges from 1 to 100,000 and the 7 axis ranges from 0 to 1.2. All lines generally trend up except for 40C at 1 pg/mL. 35C is generally above 37C and 37C is generally above 40C. There are two stars above 35C at 10,000 pg/mL and there is one star above 35C at 100,000 pg/mL.

Figure 5. SEAP for cells incubated with a 1:1 mixture of LPS and NIPAM

(n = 3, *p<0.05, **p<0.01, ***p<0.001) SEAP increases with concentration of LPS and NIPAM as expected. Cells incubated at 35℃ show significantly higher SEAP than those incubated at 37℃ and 40℃ at 10,000 pg/mL and 100,000 pg/mL LPS and NIPAM

Graph shows absorbance plotted versus concentration in pg/mL for 35C, 37C, and 40C. The x axis ranges from 1 to 100,000 and the y axis ranges from 0 to 1.2. The lines are generally flat at zero except for at 1 and 100,000 where 40C and 37C are slightly raised. 40C is above 37C. There is a star in between 40C and 35C at 1 pg/mL.

Figure 6. SEAP for cells incubated with NIPAM alone

(n = 3, *p<0.05, **p<0.01, ***p<0.001) NIPAM alone minimally activates RAW-Blue cells as expected. Cells incubated at 40℃ show significantly higher SEAP than those incubated at 35℃ at 1 pg/mL NIPAM

Graph shows absorbance for the incubation instruments "Lionheart Microscope" and "Eppendorf Incubator." The y axis ranges from 0 to 2. The "Lionheart Microscope" value is above the "Eppendorf Incubator" value. There are three stars between the two bars.

Figure 7. SEAP for cells incubated in different Instruments

(n = 3, *p<0.05, **p<0.01, ***p<0.001) SEAP is significantly greater for RAW-Blue cells incubated with 5 ng/mL LPS in the Lionheart microscope

Conclusions and Future Study

Higher incubation temperature seems to have an overall attenuating effect on LPS activation of TLR4 pathway
Difference in activation between 35℃ and 40℃ of 10,000 pg/mL and 1,000,000 pg/mL LPS and NIPAM may indicate an LCST
Cells incubated in different instruments have significantly different activations, complicating the picture

Future Research

Find new method for dual incubation to remove variables
Alter NIPAM concentration to align better with its physical properties
Cloud-point LCST of NIPAM

The following is an image of poster presented at the 2026 Undergraduate Research Forum

Acknowledgements

I would like to thank Dr. Rock Mancini and all members of the Mancini Lab for their constant support, advice, and kindness. BioRender was used to create figures 1 and 3. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number 1R01CA234115 to Dr. Rock J. Mancini. The opinions and/or findings expressed are those of the author and should not be interpreted as representing the official views or policies of the NIH or U.S. Government.

References

Hervé, C.; Laupèze, B.; Giudice, G.; Didierlaurent, A. M.; Tavares Da Silva, F. The how’s and what’s of vaccine reactogenicity. Npj Vaccines2019, 4, 39. DOI: 10.1038/s41541-019-0132-6

Shaibie, N. A.; Ramli, N. A.; Faizal, N. D. F. M.; Srichana, T.; Amin, M. C. I. M. Poly(N-isopropylacrylamide)-Based Polymers: Recent Overview for the Development of Temperature-Responsive Drug Delivery and Biomedical Applications. Macromolecular Chemistry and Physics2023, 224(20), 2300157. DOI: 10.1002/macp.202300157

Lanzalaco, S.; Armelin, E. Poly(N-isopropylacrylamide) and Copolymers: A Review on Recent Progresses in Biomedical Applications. Gels2017, 3(4), 36. DOI: 10.3390/gels3040036.

Saitoh, T.; Hamazono, Y.; Sakurai, T.; Matsubara, C. Extraction of lipopolysaccharides from water using a thermoresponsive polymer. Bunseki Kagaku2000, 49(8), 631-633. DOI: 10.2116/bunsekikagaku.49.631

Hendricksen, A. T.; Ezzatpour, S.; Pulukuri, A. J.; Ryan, A. T.; Flanagan, T. J.; Frantz, W.; Bucholz, D. W.; Ortega, V.; Monreal, I. A.; Sahler, J. M.; Nielsen, A. E.; Aguilar, H. C.; Mancini, R. J.; Thermophobic Trehalose Glycopolymers as Smart C-Type Lectin Receptor Vaccine Adjuvants. Advanced Healthcare Materials2023, 12(19), 2202918, DOI: 10.1002/adhm.202202918

NACE Career Readiness Competencies

Through my research experience, I have developed critical thinking and teamwork skills and have practiced communication through this presentation and others.

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