Lymphatic-draining nanoparticles deliver Bay K8644 payload to lymphatic vessels and enhance their pumping function

Abstract

Dysfunction of collecting lymphatic vessel pumping is associated with an array of pathologies. S-(-)-Bay K8644 (BayK), a small-molecule agonist of L-type calcium channels, improves vessel contractility ex vivo but has been left unexplored in vivo because of poor lymphatic access and risk of deleterious off-target effects. When formulated within lymph-draining nanoparticles (NPs), BayK acutely improved lymphatic vessel function, effects not seen from treatment with BayK in its free form. By preventing rapid drug access to the circulation, NP formulation also reduced BayK's dose-limiting side effects. When applied to a mouse model of lymphedema, treatment with BayK formulated in lymph-draining NPs, but not free BayK, improved pumping pressure generated by intact lymphatic vessels and tissue remodeling associated with the pathology. This work reveals the utility of a lymph-targeting NP platform to pharmacologically enhance lymphatic pumping in vivo and highlights a promising approach to treating lymphatic dysfunction.

Fig. 1.. NPs provide an LV-targeting advantage compared to free drug and allow for efficient loading and controlled release of small-molecule Bay K.

(A) Schematic of BayK’s effect on LV pumping. (B) Appearance of injected dye in tail collecting LVs (left) after coinjection with a lymphatic-draining PEG tracer (right). Scale bar, 3 mm. (C) Schematic of Pluronic-stabilized PPS NP structure and loading with hydrophobic small-molecule BayK. (D) Absorbance spectra of BayK and NPs. (E) Elution profiles of BayK and NP vehicle from a CL-6B size exclusion column when run separately or (F) after brief mixing. NP signal in each fraction was monitored using a modified iodine assay, and BayK presence was determined by measuring each fraction’s absorbance at 415 nm. (G) NP hydrodynamic diameter before and after BayK loading, measured by light scattering. (H) In vitro release profile of BayK from BayK-NP at room temperature (RT) and 37°C (n = 3). Loss of free BayK through the membrane is also shown. (I) BayK encapsulation efficiency when mixed with NP (30 mg/ml). a.u., arbitrary units.

Fig. 2.. Effect of BayK-NPs on ex vivo pumping of isolated rat mesenteric LVs.

Contraction frequency (A), contraction amplitude (B), and ejection fraction (C) by LVs isolated from the rat mesentery after exposure to BayK. Asterisk indicates statistically significant difference compared to pretreatment 100% function by one-way repeated-measures analysis of variance (RM ANOVA) (n = 8). (D) Representative recordings of spontaneous contractions in an isolated LV ex vivo before and after treatment with 200 nM BayK, (E) 200 nM BayK-NP, or (F) dose-matched control NP. (G) Change in vessel contraction frequency, amplitude, and ejection fraction after treatment with NP, 200 nM BayK-NP, or 200 nM BayK. Presented as normalized to pretreatment baseline. Asterisk indicates significant difference between treatment value and a pretreatment value of 100% by t test with Holm-Sidak correction (n = 7 to 8). In all panels, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3.. NP-formulated BayK, but not free BayK, acutely improves lymphatic pumping in vivo.

(A) Representative NIR image of LVs and resulting fluorescent intensity trace. Vessels are imaged from the left or right side of the tail. Regions of interest are selected on visible vessels, and fluorescent intensity traces are analyzed to obtain pumping metrics. Scale bar, 2 mm. (B) Packet frequency, (C) normalized packet amplitude, (D) normalized packet integral, and (E) normalized packet transport immediately after (0 hours), 8 hours after, and 15 hours after tail tip injection of BayK, BayK-NP, or their vehicle controls (DMSO and NP, respectively); all metrics presented as separate left and right vessels normalized to respective vehicle controls. Outliers were identified by ROUT (robust regression and outlier removal, Q = 1%) and removed before analysis. Significance tested by unpaired t tests between BayK formulation and appropriate vehicle control. In all panels, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.. BayK-NP show reduced side effects and toxicity compared to free drugABCDEF

(A) Concentration of BayK detected in blood after tail injection of BayK-NP or free BayK. Asterisk indicates significant difference by t test. (B) Side effect severity scale. (C) Quantification of observed side effects. Asterisk indicates significant difference by t test. (D) AST and (E) ALT activity in serum. In (D) and (E), differences were evaluated by one-way ANOVA with Tukey’s post hoc analysis (n = 4 to 10). (F) Spleen mass after chronic BayK-NP treatment. Differences tested by t test (n = 10). In all panels, *P < 0.05 and**P < 0.01. ns, not significant.

Fig. 5.. The single vessel ligation lymphedema model shows dysfunction in intact LVs.

(A) Schematic of surgical induction of lymphedema. (B) Surgery allows flow though the intact, but not ligated, LVs. Red arrow, ligation; yellow arrow, no flow past ligation; green arrow, dye accumulation distal to ligation; blue arrow, dye flow past ligation. Scale bar, 2 mm. (C) Representative tail images on d0 (before surgery), d7, and d14. (D) Tail diameter distal to ligation in individual mice following surgery. Asterisk indicates difference from d0 by one-way RM ANOVA. (E) Vessel function after surgery. One pumping pressure outlier was identified by ROUT (Q = 1%) and removed before analysis. Asterisk indicates difference from d0 100% function by one-way RM ANOVA. (F) Vessel function metrics correlated with tail diameter. (G) Fluorescence signal in the tail after tail tip dextran or NP injection, quantified from IVIS images. Asterisk indicates difference between no disease WT and lymphedema (lymph) surgery animals by two-way RM ANOVA. (H) Representative IVIS images of tails after 30-nm dextran injection. (I) Dextran and NP accumulation in individual sacral LNs in WT and lymphedema surgery mice. Asterisk indicates difference by one-way ANOVA with Tukey’s comparison. (J) NIR imaging of 680-NP uptake into LVs on d14, top view. Blue arrow, NP flow past ligation site; green arrow, distal accumulation. Scale bar, 2 mm. In all panels, *P < 0.05 and **P < 0.01.

Fig. 6.. BayK-NP treatment protects against lymphatic pumping pressure failure in lymphedema.

(A) BayK side effects in lymphedema mice immediately after treatment were significantly reduced by NP formulation. (B) Mice treated with BayK-NP had significant improvement in pressure generation 14 days after injury compared to treatment with free BayK. (C) A greater proportion of mice maintained measurable pumping efficacy 14 days after injury when treated with BayK-NP rather than free BayK. Statistical significance determined by Fisher’s exact test comparing BayK-NP effect to combined effect of control groups. (D) Quantification of collagen⁺ area between the muscle fascia and dermis for BayK treatments normalized to respective vehicle control. Number sign indicates significantly lower collagen deposition relative to vehicle control by t test against normalized control; asterisk indicates significantly stronger effect relative to vehicle control than observed from free BayK formulation. (E) Sirius Red staining for collagen in mouse tail sections 14 days after lymphedema induction. Scale bars, 500 μm. In all panels, *P < 0.05 and **P < 0.01.