Therapeutic Polymer Conjugates and Their Characterization

Abstract

Drugs, proteins, and nanoparticles delivered into the body are exposed to numerous mechanisms for premature elimination and deterioration within the body. Polymers can be covalently bound to this cargo as either bioactive or inert carriers of such therapeutics to impart protective characteristics. These include improved biodistribution, first-pass clearance reduction, specific targeting, and immune evasion. These polymers can be either synthetic or naturally derived and degradable or inert. Both the polymer and the cargo of interest may be joined together using a series of controlled and specific conjugation chemistries that target functional groups on either species to form covalent bonds. In addition, selectively cleavable linkers may be utilized to engineer cargo release at specific locations under unique physiological conditions such as low pH, oxygen, or biomolecule-laden environments. We will discuss not only different conjugation chemistry strategies and how they should be considered for targeted applications but also how one might reconcile them with different desired downstream characterization as well as in vivo uses.

Graphical Abstract

Quiz Questions (Multiple Choice)

• Question 1: A positively charged hydrophilic PEG-small molecule dendrimer is found to have a hydrodynamic radius of 3 nm. Which of the following organs would play the biggest role in clearing the conjugate from a patient’s systemic circulation?

  • A. Liver.

  • B. Spleen.

  • C. Kidney.

  • D. Lungs.

• Answer: C. Kidneys have a size cutoff range of 6-8 nm. Factors such as charge can influence glomerular filtration as well.

• Question 2: A protein-peptide conjugate is created to sterically block an immunogenic epitope on the protein. Thus, its diffusivity is similar to a protein of about 10⁻⁶ cm²/s. What would be the expected hydrodynamic radius of such a particle in water?

  • A. 2.27 x10⁻⁹ m.

  • B. 2.88 x10⁻⁸ m.

  • C. 2.13 x10⁻¹³ m.

  • D. 2.16 x10⁻⁶ m.

• Answer: A. First, the diffusivity of a protein is converted to m²/s. Using the Boltzmann constant of 1.38 x10⁻²³ m²*kg*s⁻²*K⁻¹, an average body temperature of 310 K, the viscosity of water (10⁻³ Pa) can all be plugged into the Stokes-Einstein equation. After converting the units, the denominator yields a value of 1.884x10⁻¹² leaving all units cancelled while the numerator gives 4.278x10⁻²¹ m. Dividing these two values gives the final answer of 2.27x10⁻⁹ m or 2.27 nm radius.

• Question 3: Your company is testing a new experimental branched pHPMA-drug conjugate that has hydrolytically degradable linkers along its backbone for controlled release of the drug. Which of the following purification modalities would likely NOT be suitable for your prototype therapeutic?

  • A. Dialysis.

  • B. Centrifugation.

  • C. Size exclusion chromatography.

  • D. Gel electrophoresis.

• Answer: A. Dialysis uses a semipermeable membrane to filter out unreacted components of a reaction using water. A hydrolytically degradable bond has the potential to be denatured or cleaved during this process.

  Question 4: Your research lab hopes to create an amphiphilic micelle structure to increase the solubility of a hydrophobic drug. Which of the following assays would be most useful to characterize the shape of the conjugates. Additionally, which values indicate a spherical particle?

  Dynamic Light scattering, R_g/R_H > 0.775.

  • A. Small angle x-ray diffraction and dynamic light scattering, R_g/R_H < 0.775.

  • B. Small angle x-ray diffraction, R_g/R_H > 0.775.

  • C. Small angle x-ray diffraction and dynamic light scattering, R_g/R_H > 0.775.

• Answer: B. DLS gives hydrodynamic radius R_H while small angle x-ray diffraction gives R_g. Thus, both would be needed. The ratio of R_g/R_H for a globular (circular) particle is less than 0.775.

• Question 5: A 100 Da PEG chain has a monomer length of 3.5 Å. If a nanoparticle has a radius of 10 nm. The distance between PEG monomers is 100 Å. which of following confirmations would the pegylated nanoparticle take?

  • A. Mushroom.

  • B. Brush.

  • C. The particle will have both mushroom and brush confirmations.

  • D. Not enough information.

• Answer: A. Using the equation for the Flory Radius yields 55.47 Å. Since the R_F is lower than 100 Å, the confirmation of the coating will likely be mushroom.

• Question 6: You are designing a drug that disrupts the metabolic pathway of a cancer cell and have decided to use a degradable linker to facilitate intracellular release. Which of the following is the least suitable for this task?

  • A. Hydrazide (pH).

  • B. Oligo-peptide chain (enzyme).

  • C. Ester bonds (hydrolytic).

  • D. Disulfide bond (reduction).

• Answer: C. Engineered intracellular release can be facilitated by using bonds sensitive to cleavage of enzymes, lower pH, and a reducing environment. Hydrolysis is a less ideal method due to potential degradation in systemic circulation.

• Question 7: A dendrimer contains encapsulated Doxorubicin (DOX) and you’re tasked with studying the release of the drug over time. Doxorubicin is fluorescent when it leaves the nanoparticle. The polymer has the composition such that it is comprised of hydrolytically cleavable ester bonds. You want to prevent burst release of DOX from these nanoparticles. Which of the following rates would best represent such as release?

  • A. τ_reaction > τ_diffusion.

  • B. τ_reaction < τ_diffusion.

  • C. τ_reaction = τ_diffusion.

  • D. there is no difference in outcomes.

• Answer: B. Burst release can be characterized by a rapid cleavage of the hydrolytically degradable bond such that the rate (τ_reaction) is larger than diffusion (τ_diffusion). Thus, for slower release of the drug, τ_reaction < τ_diffusion.

• Question 8: You’re studying different molecular weight PEG chains to increase the hydrodynamic radius of a chemotherapeutic. However, with increased retention time, your tasked to test possible toxicity relating to these new formulations. Which of the following assays would NOT be suitable?

  • A. Live/dead viability in vitro.

  • B. MTT assay.

  • C. ATS/ALS ELISA.

  • D. Live fluorescent imaging.

• Answer: D. Live fluorescent imaging is useful for tracking a particle in vivo in real time. It would not be ideal to investigate toxicity.

• Question 9: Which of the following are NOT considerations when conjugating proteins?

  • A. Reduction of functionality due to steric hindrance.

  • B. Similar molecular weight of polymer chains leading to difficulty characterizing.

  • C. Linkers must be used to bind the initiator to the functional group.

  • D. Neighboring amino acid residues may lead to off targeting chemistries.

• Answer: C. Linkers do not necessarily have to be used in protein conjugation strategies. Polymers may be covalently bound to proteins using a functional amino acid residue and a functional group on the polymer and or initiator.

• Question 10: You are researching possible amino acid residues to target on your protein for conjugation to a biomaterial carrier. You determine that you need a site-specific reaction for this. Which of the following would NOT be appropriate?

  • A. Reductive amination of a lysine group.

  • B. Disulfide exchange of a cysteine.

  • C. Recombination of the protein to introduce a non-canonical amino acid.

  • D. Enzymatic ligation of a C-terminus.

• Answer: A. Lysine is generally the most abundant amino acid on the surfaces of proteins. Thus, conjugation strategies targeting lysines are usually performed for the purpose of speckled coatings and not for epitope or site-specific reactions.