Theranostic Design of Angiopep-2 Conjugated Hyaluronic Acid Nanoparticles (Thera-ANG-cHANPs) for Dual Targeting and Boosted Imaging of Glioma Cells

Simple Summary Glioblastoma multiforme is the most aggressive malignant brain tumor with poor patient prognosis. The presence of the blood-brain barrier and the complex tumor microenvironment impair the efficient accumulation of drugs and contrast agents, causing late diagnosis, inefficient treatment and monitoring. Functionalized theranostic nanoparticles are a valuable tool to modulate biodistribution of active agents, promoting their active delivery and selective accumulation for an earlier diagnosis and effective treatment, and provide simultaneous therapy and imaging for improved evaluation of treatment efficacy. In this work, we developed angiopep-2 functionalized crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and irinotecan (Thera-ANG-cHANPs) that were shown to boost relaxometric properties of Gd-DTPA by the effect of Hydrodenticity, improve the uptake of nanoparticles by the exploitation of angiopep-2 improved transport properties, and accelerate the therapeutic effect of Irinotecan. Abstract Glioblastoma multiforme (GBM) has a mean survival of only 15 months. Tumour heterogeneity and blood-brain barrier (BBB) mainly hinder the transport of active agents, leading to late diagnosis, ineffective therapy and inaccurate follow-up. The use of hydrogel nanoparticles, particularly hyaluronic acid as naturally occurring polymer of the extracellular matrix (ECM), has great potential in improving the transport of drug molecules and, furthermore, in facilitatating the early diagnosis by the effect of hydrodenticity enabling the T1 boosting of Gadolinium chelates for MRI. Here, crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and the chemotherapeutic agent irinotecan (Thera-cHANPs) are proposed as theranostic nanovectors, with improved MRI capacities. Irinotecan was selected since currently repurposed as an alternative compound to the poorly effective temozolomide (TMZ), generally approved as the gold standard in GBM clinical care. Also, active crossing and targeting are achieved by theranostic cHANPs decorated with angiopep-2 (Thera-ANG-cHANPs), a dual-targeting peptide interacting with low density lipoprotein receptor related protein-1(LRP-1) receptors overexpressed by both endothelial cells of the BBB and glioma cells. Results showed preserving the hydrodenticity effect in the advanced formulation and internalization by the active peptide-mediated uptake of Thera-cHANPs in U87 and GS-102 cells. Moreover, Thera-ANG-cHANPs proved to reduce ironotecan time response, showing a significant cytotoxic effect in 24 h instead of 48 h.

Microfluidic Set Up -Thera -cHANPs To efficiently encapsulate Irinotecan, a cosolvation strategy is explored in order to overcome limitations due to the low water solubility of Irinotecan (1 mg/mL) (the procedure is reported in the dedicated method section). After production, nanoparticles are gently concentrated via Rotary evaporation in order to reach therapeutic concentrations of Irinotecan. SEM images of both Thera cHANPs and concentrared Thera-cHANPs are reported to demonstrate the stability of NPs against concentration procedure. c) Figure S2 -SEM images of a) Thera-cHANPs; b) PSD of Thera-cHANPs; c) concentrated Thera-cHANPs.

Angiopep-2 bioconjugation reaction optimization
Quantity of peptide Different concentrations of Angiopep-2 were added to cHANPs suspension and different times of contact were tested in order to assess the best reaction condition. The amount of peptide bound to nanoparticles exposed at 20 ug/mL of Angiopep-2 after 3h is reported equal to 0, which means that in this measurement, all the absorbances of the triplicate were comparable or slightly lower than the absorbance of bare cHANPs, so that the reported value is equal to zero.

a) b) Intensity
Purification method Ultracentrifugation (UC) Via UC, it is not possible to efficiently sediment nanoparticles and remove the excess of peptide without a significant loss of nanoparticles. DLS measurements of sediment and supernatant show similar size distributions ( Figure S1), attenuator values and counts per second (kps) revealing similar nanoparticle concentration in both samples, thus inefficient separation. Figure S2 shows the effect of rpm and centrifugation time in CC of both 30 and 50 kDa MWCO on the volume of the concentrated phase. SEM observations of the concentrated and continuous phase of all the tested conditions allow us to define as optimized purification condition 3000 rpm for 10 min with a 50 kDa filter. In this condition, no aggregation in the concentrated phase and a minimum loss in the continuous phase are observed, Figure S3.     NPs Imaging properties MRI images of NPs were acquired on a 1T SPECT/MRI system (NanoScan; Mediso Medical Imaging) with a solenoid coil with 35mm diameter in 30 min. Multiple T1 images were acquired using a spin-echo sequence (repetition times 550, 880, 1400, 2000 ms, echo time 80 ms) and T1 maps were calculated using MLE method to fit saturation recovery curves (a*exp(-TR/T1)+c). The other scan parameters included an 80-mm field of view, a 96 × 96 matrix, and 2 mm slice thickness.

Characterization of payload agents in Co-loaded ANG-cHANPs
T1 weighted images at 37°C by a 1 Tesla clinical MRI scanner are acquired to compare cHANPs at different concentration co-loading Gd-DTPA and Irinotecan. In the following figure a T1 map scale bar is also reported to observe the difference in the relaxation time. Starting from the top-right image and moving anticlockwise (from 1 to 4) , it is possible to observe a reduction in the shortening of the T1 signal due to a lower concentration of the Gd-DTPA obtained by the diluition of the cHANPs measured at concentrations from 100 µM to 12,5 µM. Please note that the range of measured concentrations has been chosen to stay in the linear range of relaxivity. These results confirm the ability of the cHANPs to be also used in MRI.

Figure S7 -Stability of cHANPs in culture medium at 37°C
Short times uptake rates of cHANPs and ANG-cHANPs by U87 cells Figure S8 -Comparison of uptake rates by U87 cells of cHANPs and ANG-cHANPs in short times Figure S9 -MTT assay on U87-MG cells incubated with 10 µM of free Irinotecan, Irinotecan in Thera-cHANPs and in Thera-ANG-cHANPs. Raw Flow Cytometry data of Thera-cHANPs on U87 cells