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. 2020 Feb 10;59(7):2776-2783.
doi: 10.1002/anie.201911762. Epub 2020 Jan 7.

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing

Natalie Boehnke  1 , Santiago Correa  1   2   3 , Liangliang Hao  1   4 , Wade Wang  5 , Joelle P Straehla  1   6 , Sangeeta N Bhatia  1   4   7   8   9 , Paula T Hammond  1   10
Affiliations

Theranostic Layer-by-Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing

Natalie Boehnke et al. Angew Chem Int Ed Engl. .

Abstract

Layer-by-layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer-by-layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary-based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.

Keywords: gene delivery; layer-by-layer assembly; nanoparticles; self-assembly; theranostics.

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Conflict of interest statement

Conflict of Interest:

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Click-functionalized LbL NPs remain stable and exhibit protease-responsive behavior.
(a) Diameter, polydispersity index, and (b) zeta potential of click-modified LbL NPs. (c) In vitro cleavage of biosensor LbL NPs in the presence of MMP9. Nanoparticle modifications are abbreviated as follows: biosensor peptide (+S), iRGD targeting peptide (+R), and PEG pendants (+P). Error bars represent standard deviation, and statistical analysis of panel b uses one-way ANOVA with a Dunnett post-hoc test and alpha of 0.05.
Figure 2.
Figure 2.. Addition of the biosensing peptide onto siRNA-loaded LbL NPs provides a NP capable of simultaneous tumor detection and gene silencing.
Analysis of the urine of the mice one hour after injection indicates that theranostic LbL NPs yield significantly elevated levels of the peptide reporter fragment in the urine in a (a) pancreatic cancer flank model, (b) a metastatic model of colorectal cancer, and (c) two models of ovarian cancer. (d) Theranostic LbL NPs were intravenously administered via tail vein injection to the subcutaneous flank ovarian cancer model to provide an siRNA dosage of 0.5 mg/kg to silence the model gene luciferase in vivo. (e) Bioluminescence levels were monitored over three days using an IVIS imaging device, also used to generate the images shown in a–c. Error bars represent SEM, and statistical analysis of panels a and b uses students t-tests, while panel c uses a one-way ANOVA with a Dunnett post-hoc test and alpha of 0.05. Panel e uses a one-way ANOVA with a Tukey post-hoc test and alpha of 0.05.
Figure 3.
Figure 3.. Addition of targeting ligands and biosensor peptides improves LbL NP binding to ovarian cancer cells.
Flow cytometry was used to assess NP-associated fluorescence of OVCAR8 cells after incubating with NPs for (a) 4 and (b) 24 hours. Analysis of the mean NP-associated fluorescence intensity of the NP-positive cell population at (c) 4 and (d) 24 hours. Error bars represent SEM, and statistical analysis was carried out using a one-way ANOVA with Tukey’s post-hoc test and an alpha of 0.05. (e) Super resolution microscopy images of OVCAR8 cells incubated with sulfoCy-3 labeled LbL NPs for 24 hours. Wheat germ agglutinin, shown in red, was used to stain endosomal membranes. NPs are shown in green and nuclei in cyan. Scale bar = 10 μm.
Figure 4.
Figure 4.. Theranostic LbL NP biodistribution in a metastatic model of ovarian cancer indicates tumor targeting capabilities, especially using intraperitoneal (IP) administration.
(a) A metastatic model of ovarian cancer was used to evaluate whether IP-administration of nanoparticles improved biodistribution and sensistivity of the diagnostic function of the theranostic NPs. (b) NP accumulation in liver, spleen, and tumors was measured 72 hours post-administration using a fluorescent flatbed scanner (Li-COR Odyssey). Tissue autofluorescence was used to generate a region-of-interest (indicated by the white outline) in which to quantify signal from the Cy7-labeled NPs (Figure S6). (c) Urine was collected 1-hour post-IP administration of NP+SR and analyzed for the MMP9-sensitive biomarker. (d–f) Cryohistology was performed on tumor tissue and imaged using a fluorescent slide scanner to detect tissue autofluorescence (pseudo-colored magenta) and NP signal (pseudo-colored green). These representative images of LbL NP distribution through tumors when administered (d–e) IP and (f) intravenously. Scale bar = 100 μm. Error bars represent SEM, and statistical analysis was carried out using an unpaired student’s t-test.
Scheme 1.
Scheme 1.
Biosensor peptides can be conjugated onto siRNA-containing LbL liposomes via copper(I)-catalyzed click chemistry to create theranostic LbL NPs. In the tumor microenvironment, biosensor peptides are cleaved, and the resulting fragments can be non-invasively detected in the urine to monitor disease progression. PLR = poly-l-arginine, pPLD = propargyl-modified poly-l-aspartic acid..
See this image and copyright information in PMC

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