Effect of repeated NiNLP administration on mouse organ weights.

<p>Weights of A) liver, B) kidney, C) lung, and D) spleen obtained from mice that received 25 µg of NiNLP i.n. (30 µl) or i.p. (100 µl) daily for 14 consecutive days. Control animals received an equal volume of PBS i.p.(100 µl) daily for 14 days. Normalized organ weights are represented as (organ weight, g)/(body weight, g). Data represent averaged organ weights from groups of three animals, with standard deviation error bars.</p

Stability of DMPC∶NLPs in 20% serum assessed by SEC over 24 hrs at 25°C.

<p>The indicated peaks correspond to intact NLP (t_R 9.5 min) and free apoE422k scaffold protein (t_R 12.2 min). Absorbance of AF647-labeled apoE422k absorbance was monitored at 600 nm.</p

Stability of NLPs as a function of lipid content, temperature, time, and serum concentration.

<p>Integrated NLP peak area of the SEC chromatograms for A) DOPC∶NLPs incubated at 25°C and B) DMPC∶NLPs incubated at 25°C. C) t_1/2 of the DOPC∶NLPs (blue line) and DMPC∶NLPs (red line) incubated at 25°C. Integrated NLP peak area of the SEC chromatograms for D) DOPC∶NLPs incubated at 37°C and E) DMPC∶NLPs incubated at 37°C. F) t_1/2 of the DOPC∶NLPs (blue line) and DMPC∶NLPs (red line) incubated at 37°C. AF647-labeled apoE422k absorbance was monitored at 600 nm.</p

Time-dependent <i>in vivo</i> NiNLP biodistribution upon i.p. and i.n. administration.

<p>NiNLPs were administered by A) i.p. or B) i.n. routes and were assessed over 72 or 96 hours, respectively. Organ fluorescence was determined <i>ex vivo</i> and normalized to total organ weight. The normalized fluorescent intensity was quantitatively measured as a function of time. Data represent the average normalized fluorescence from groups of two animals, with standard error bars.</p

Effect of repeated NiNLP administration on mouse body weights.

<p>Weights of A) male and B) female mice receiving daily NiNLP injections i.n. (30 µl) or i.p. (100 µl) for 14 consecutive days. Control mice received equal volumes of PBS i.p. over the same 14-day time course. Data represent averaged weights from groups of three animals, with standard deviation error bars.</p

Non-covalent conjugation of protein (0841) to the NiNLP platform.

<p>A) SEC traces of NiNLPs incubated with His-tagged 0841 at indicated molar ratios. B) The 0841:NiNLPs were purified by SEC, and SDS-PAGE and densitometry were used to quantify the amount of 0841 and apoE422k protein in each NiNLP sample using known standards for each protein. These measured concentrations were used to calculate the number of proteins bound per NLP and were plotted as a function of the initial ratio used in the conjugation reaction.</p

Histological analysis of the effect of repeated NiNLP administration on the microstructure of the liver.

<p>H&E stained sections of livers obtained from mice that had received 25 µg of NiNLP i.n. (30 µl) or i.p. (100 µl) daily for 14 consecutive days at 10×, 20× and 40× magnifications.</p

Covalent conjugation of protein (0841) to the N₃NLPs platform.

<p>A) and B) SEC chromatograms of 0841 (black line) and 0841-DBCO (blue line) incubated with AF488-azide at a fluorescent dye to protein ratio of 5 monitored at two absorbance wavelengths: A) 280 nm and B) 495 nm. Chromatogram peaks correspond to 0841 protein (t_R 12.5 min) and unreacted AF488-azide (t_R 16.3 min). C) SEC chromatograms of N₃NLPs incubated with 0841-DBCO at indicated molar ratios. Chromatogram peaks correspond to NLP (t_R 8.5 min) and unconjugated 0841 protein (t_R 12.5 min). D) The 0841-DBCO:N₃NLPs complexes were purified by SEC, and SDS-PAGE and densitometry were used to quantify the amount of 0841 and apoE422k protein in each NiNLP sample using known standards for each protein. These measured concentrations were used to calculate the number of proteins bound per NLP and were plotted as a function of the initial ratio used in the conjugation reaction. E) SEC chromatograms of NLPs (lacking the C18-PEG6-N₃) incubated with 0841-DBCO at indicated molar ratios. F) SEC chromatograms of N₃NLPs incubated with 0841 (lacking DBCO reactive group) at indicated molar ratios.</p

Assessment of NiNLP immunogenicity.

<p>Groups of 10 female BALB/c mice were inoculated either i.n. or i.p. with NiNLP. As a positive control, a group of mice was injected with a known immunogenic recombinant subunit antigen (LcrV) co-administered with adjuvant (CpG). Serum IgG antibody titers against the scaffold protein, apoE422k (NiNLP-ip and NiNLP-in), or LcrV (LcrV+CpG-ip and LcrV+CpG-in) were assessed 4 weeks post-immunization. Each data point represents the titer value of an individual mouse.</p

Conjugation of cODN and PF to the NLP platform.

<p>A) SEC analysis of the cODN∶NLP constructs at indicated cODN∶NLP molar ratios, monitored at 280 nm. The increase in absorbance at 280 nm and peak shift indicate successful incorporation of cODN. B) UV-Vis absorption spectra of SEC-purified cODN∶NLP (blue line) and apoE422k concentration matched NLP lacking the cODN (black line). The dashed lines represent the absorbance at 260 nm and 280 nm. C) Analysis of cODN incorporation into the NLP. The x-axis represents the cODN∶NLP ratio used during the NLP assembly reaction and the y-axis is the measured amount of cODN ultimately incorporated into the particle. D) SEC chromatograms of the PF∶NLP constructs at increasing PF-to-NLP ratios. E) UV-vis spectra of PF∶NLPs (blue line) and apoE422k concentration matched NLPs lacking the PF (black line). The dashed lines represent the absorbance at 280 nm and 368 nm. F) Analysis of PF incorporation efficiency into the NLP, represented as a function of the PF-to-NLP assembly ratio (x-axis) vs. measured PF-to-NLP ratio after purification (y-axis).</p