Hyaluronic Acid Molecular Weight Determines Lung Clearance and Biodistribution after Instillation

Abstract

Hyaluronic acid (HA) has emerged as a versatile polymer for drug delivery. Multiple commercial products utilize HA, it can be obtained in a variety of molecular weights, and it offers chemical handles for cross-linkers, drugs, or imaging agents. Previous studies have investigated multiple administration routes, but the absorption, biodistribution, and pharmacokinetics of HA after delivery to the lung is relatively unknown. Here, pharmacokinetic parameters were investigated by delivering different molecular weights of HA (between 7 and 741 kDa) to the lungs of mice. HA was labeled with either a near-infrared dye or with iodine-125 conjugated to HA using a tyrosine linker. In initial studies, dye-labeled HA was instilled into the lungs and fluorescent images of organs were collected at 1, 8, and 24 h post administration. Data suggested longer lung persistence of higher molecular weight HA, but signal diminished for all molecular weights at 8 h. To better quantitate pharmacokinetic parameters, different molecular weights of iodine-125 labeled HA were instilled and organ radioactivity was determined after 1, 2, 4, 6, and 8 h. The data showed that, after instillation, the lungs contained the highest levels of HA, as expected, followed by the gastrointestinal tract. Smaller molecular weights of HA showed more rapid systemic distribution, while 67 and 215 kDa HA showed longer persistence in the lungs. Lung exposure appeared to be optimum in this size range due to the rapid absorption of <67 kDa HA and the poor lung penetration and mucociliary clearance of viscous solutions of HA > 215 kDa. The versatility of HA molecular weight and conjugation chemistries may, therefore, provide new opportunities to extend pulmonary drug exposure and potentially facilitate access to lymph nodes draining the pulmonary bed.

Keywords: biodistribution; haluronic acid; pharmacokinetics; pulmonary delivery; pulmonary transport; radiolabeled.

Figure 1

Depiction of full HA monomer unit, abbreviated HA, DMTMM molecule, and IR dye as a hydrazine group. (A) Chemical reaction of HA with DMTMM and tyrosine for tyrosine conjugation followed by addition of ¹²⁵I for radiolabeled HA. (B) Hydrazine IR dye reaction leading to conjugation on HA.

Figure 2

Fluorescent images of lungs injected with HA-IR showing distribution though out all lung lobes with all five different molecular weights. (A) Lungs injected with 5 µL of 7 kDa HA solution; (B) 7 kDa HA 8 h; (C) 30 kDa HA 8 h; (D) 67 kDa HA 8 h; (E) 215 kDa HA 8 h; (F) 741 kDa 8 h. White scale bar is 1 cm. The white numbers represent lobes of the lungs with 1, 2, and 3 representing mouse anatomical right lung lobes and 4 and 5 representing mouse anatomical left lung lobes.

Figure 3

Analysis of lungs instilled with HA–IR dye. The largest molecular weight of HA had the longest persistence in the lungs which decreased with molecular weight. ** signifies statistical difference from 7, 30, and 67 kDa HA, and each point is the average of at least three animals.

Figure 4

Exposure levels of HA in the lungs averaged across both right and left lung lobes as a percentage of delivered dose over time for each of the five HA molecular weights. Each point is an average of at least five animals with the error on each point <5% of the measurement. * represents p value <0.05 for 67 kDa and 215 kDa HA compared with 7 kDa, 30 kDa, and 741 kDa HA. ** represents p value <0.05 for 30 kDa and 741 kDa HA compared with 7 kDa, 67 kDa, and 215 kDa HA. *** represents p value <0.05 for 30 kDa, 67 kDa, 215 kDa, and 741 kDa HA compared with 7 kDa HA.

Figure 5

Exposure levels of HA in the stomach as a percentage of delivered dose over time for each of the five HA molecular weights. Each point is an average of at least five animals with the error on each point <5% of the measurement. * represents p value <0.05 for 741 kDa HA compared with 7 kDa, 30 kDa, 67 kDa, and 215 kDa HA.

Figure 6

Exposure levels of HA in the intestine as a percentage of delivered dose over time for each of the five HA molecular weights. Each point is an average of at least five animals with the error on each point <5% of the measurement.

Figure 7

Exposure levels of HA in the heart, spleen, liver, kidney, bladder, and trachea based on molecular weight with 7 and 29 kDa HA on top, 67 and 215 kDa HA in the middle, and 741 kDa HA on the bottom as a percentage of delivered dose over time for each of the five HA molecular weights. Each point is an average of at least five animals with the error on each point <5% of the measurement.