ENGINEERING
`
`APPLIEDBIOLOGICAL
`
`SCIENCES
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`Nanoconjugation modulates the trafficking and
`mechanism of antibody induced receptor endocytosis
`
`Sanjib Bhattacharyyaa, Resham Bhattacharyaa, Steven Curleyb, Mark A. McNivena,c, and Priyabrata Mukherjeea,d,c,1
`
`aDepartment of Biochemistry and Molecular Biology, dDepartment of Biomedical Engineering, cCancer Center Mayo Clinic, Rochester, MN 55905; and
`bDepartment of Surgery, MD Anderson Cancer Center, Houston, TX
`
`Edited* by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved July 7, 2010 (received for review May 11, 2010)
`
`Treatment with monoclonal antibody (mAbs) is a viable therapeutic
`option in cancer. Recently, these mAbs such as cetuximab, hercep-
`tin, etc., have been used as targeting agents to selectively deliver
`chemotherapeutics to cancerous cells. However, mechanisms of
`nanoparticles-mAbs interactions with the target cells and its effect
`on intracellular trafficking and mechanism are currently unknown.
`In this paper, we demonstrate that the distinct patterning and
`dynamics of anti-EGFR (epidermal growth factor receptor) anti-
`body cetuximab (C225)- induced EGFR internalization in pancreatic
`cancer cells with variable receptor expression is altered upon nano-
`conjugation. Nanoconjugation uniformly enhanced C225-induced
`EGFR endocytosis in PANC-1, AsPC-1, and MiaPaca-2 cells, influ-
`enced its compartmentalization and regulated the involvement
`of dynamin-2 in the endocytic processes. Receptor endocytosis
`and its intracellular trafficking were monitored by confocal micro-
`scopy and transmission electron microscopy. The role of dynamin-2
`in EGFR endocytosis was determined after overexpressing either
`wild-type dynamin-2 or mutant dynamin-2 in pancreatic cancer
`cells followed by tracking the receptor-antibody complex interna-
`lization by confocal microscopy. Significantly,
`these findings
`demonstrate that the nanoconjugation cannot be construed as
`an innocuous reaction involved in attaching the targeting agent
`to the nanoparticle, instead it may distinctly alter the cellular pro-
`cesses at the molecular level, at least antibody induced receptor
`endocytosis. This information is critical for successful design of a
`nanoparticle-based targeted drug delivery system for future
`clinical translation.
`
`EGFR ∣ nanoparticle ∣ dynamin ∣ pancreatic cancer
`
`Nanotechnology provides opportunities for biomedical appli-
`
`cations that may improve human health care in the future
`(1–13). Engineered nanoparticles are evolving as promising can-
`didates for various biological applications due to their tunable
`biophysical and biochemical properties (14–16). However, funda-
`mental studies to understand the mechanism of cell-nanomaterial
`interactions are still lacking. To advance the successful develop-
`ment of biomedical nanotechnology for clinical use, vivid under-
`standing of such mechanisms is essential.
`Epidermal growth factor receptor (EGFR) represents a un-
`ique target as over expression of EGFR has been implicated in
`the pathogenesis of many cancers (17, 18). Cetuximab (C225), a
`monoclonal anti-EGFR antibody, has been approved by the FDA
`for treatment of colon and head and neck cancers (19). Cetuxi-
`mab is also in different phases of clinical trials for other cancers
`(20, 21). However, the mechanism and pattern of C225 induced
`endocytosis of EGFR in pancreatic cancer cells of variable EGFR
`expression is not fully understood (20, 21). Furthermore, the
`effect of nanoconjugation on the mechanism of C225 induced
`endocytosis remains to be investigated. Here, we demonstrate
`the mechanism and endocytic pattern of C225 and gold conju-
`gated-C225 induced internalization of EGFR in pancreatic can-
`cer cell lines characterized by differential expression of EGFR
`and thus varying in metastatic potential. We also investigate
`how conjugation of cetuximab to gold nanoparticle alters its cel-
`lular mode of function in metastatic vs. primary pancreatic cancer
`
`cell lines. We demonstrate that nanoconjugation uniformly pro-
`motes faster endocytosis of EGFR, influences its compartmenta-
`lization, and regulates the involvement of dynamin-2 (dyn-2) in
`the endocytic processes. These studies provide molecular insight
`into the mechanism of C225 induced endocytosis of EGFR and
`help to design the nanoconjugates for efficient targeting.
`
`Results
`Conjugation to Gold Nanoparticles Enhances C225 Induced Endocyto-
`sis of EGFR. To test if conjugation to gold nanoparticle regulated
`the C225 induced endocytosis of EGFR in pancreatic cancer cells
`of variable EGFR expression, we tracked C225-Cy3 and Au-
`C225-Cy3 induced EGFR endocytosis by confocal microscopy.
`Previously we demonstrated the differential EGFR expression
`in PANC-1, MiaPaca2, and AsPC-1 cells (18). When cells were
`incubated with either C225-Cy3 or Au-C225-Cy3 at 4 °C for
`30 min, the majority of the C225 was primarily localized at the
`plasma membrane, suggesting an energy dependent pathway
`for endocytosis (Figs. S1, S2, and S3) (5). A distinct difference
`in the endocytosis pattern of EGFR was observed in C225-Cy3
`vs. Au-C225-Cy3 treated cells at 37 °C. In general, nanoconjuga-
`tion promoted faster endocytosis of EGFR and the pattern of
`uptake was also quite different among various pancreatic cancer
`cells with variable EGFR expression. In PANC-1 cells (primary
`cell line with high EGFR expression), C225-Cy3 was predomi-
`nantly localized at the membrane at 30 min, whereas Au-C225-
`Cy3 was predominantly visible in the perinuclear region. How-
`ever, at 1 h, EGFR (Fig. 1A, right) was completely perinuclear
`in the Au-C225-Cy3 treatment group, whereas membrane stain-
`ing could still be observed for C225-Cy3 (Fig. 1A, left). Similar
`results were obtained in MiaPaca2 cells (primary cell line with
`low EGFR expression). This data suggests that Au-C225-Cy3 pro-
`motes faster endocytosis of EGFR (Fig. 1C, right) as compared to
`C225-Cy3 (Fig. 1C, left). The pattern of EGFR endocytosis in the
`AsPC-1 cells (metastatic cell line) was distinct from PANC-1 cells
`even though they expressed similar levels of EGFR (18). In the
`case of AsPC-1 cell, Au-C225-Cy3 induced notable endocytosis
`within 1 h in the form of discrete puncta at the membrane and
`cytosol (Fig. 1B, right) as compared to PANC-1 and MiaPaca2
`cells. In contrast, C225-Cy3 was unable to induce significant
`EGFR endocytosis even after 1 h of treatment in AsPC-1 cells.
`The number of puncta formation was also significantly higher
`for Au-C225-Cy3 comparing to C225-Cy3 (Fig. 1B, left). This in-
`creased number of puncta formation suggests enhanced cluster-
`ing of EGFR induced by nanoconjugation and suggests faster
`endocytosis by nanoconjugated C225. Furthermore, we demon-
`strated previously that preincubation of various cancer cells with
`
`Author contributions: S.C., M.M., and P.M. designed research; S.B. and R.B. performed
`research; M.M. contributed new reagents/analytic tools; S.B., R.B., and P.M. analyzed data;
`and S.B., R.B., S.C., M.M., and P.M. wrote the paper.
`
`The authors declare no conflict of interest.
`
`*This Direct Submission article had a prearranged editor.
`
`1To whom correspondence should be addressed. E-mail: mukherjee.priyabrata@mayo.edu.
`
`This article contains supporting information online at www.pnas.org/lookup/suppl/
`doi:10.1073/pnas.1006507107/-/DCSupplemental.
`
`www.pnas.org/cgi/doi/10.1073/pnas.1006507107
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`PNAS ∣ August 17, 2010 ∣ vol. 107 ∣ no. 33 ∣ 14541–14546
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`Downloaded from https://www.pnas.org by 163.116.157.101 on May 31, 2023 from IP address 163.116.157.101.
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`important to note here that conjugating increasing amounts of
`C225 to gold nanoparticle did not change the pattern of endocy-
`tosis in both primary and metastatic cells, suggesting a lack of
`multivalency effect in such endocytic processes.
`Taken together, these data suggest that EGFR endocytosis
`induced by C225 binding to the receptor is distinct in primary
`(PANC-1 and MiaPaca2) vs. metastatic pancreatic cancer cells
`(AsPC-1). In addition, conjugation to gold nanoparticle enhan-
`ced C225 induced endocytosis of EGFR and alters the endocytic
`patterning of the receptor primarily to the perinuclear region in
`the case of primary cell lines whereas in the punctate form
`throughout the cytosol in the case of metastatic cell line.
`
`Conjugation to Gold Nanoparticles Altered C225 Induced Intracellular
`Trafficking of EGFR. Next we wanted to investigate if nanoconjuga-
`tion also altered the intracellular trafficking of EGFR. It is well
`documented that the ligand, EGF, induced internalization of
`EGFR leads to accumulation in the early endosomes, followed by
`the Golgi complex. Finally the receptors are either recycled back
`to the plasma membrane or degraded in the lysosomes (17, 24).
`However, the intracellular trafficking of C225 induced EGFR
`internalization (for that matter any antibody induced receptor
`endocytosis) is still not clear. To address this issue, we performed
`colocalization experiments to determine C225-Cy3 and Au-C225-
`Cy3 induced endocytosis in different organelles namely early en-
`dosomes (EEA), lysosomes, the Golgi complex, and transferrin,
`(representing the recycling compartment). In MiaPaca2 cells,
`significant colocalization of both C225-Cy3 and Au-C225-Cy3
`occurred in the early endosomes (Table 1). However, in the case
`of Au-C225-Cy3, percent colocalization with the Golgi marker
`was at least twofold higher (24.2  2.1) than the antibody itself
`(12.5  1.6), with a p value of <0.05 suggesting faster endocytosis
`(Table 1, Fig. 2A). Furthermore, colocalization with transferrin
`was significantly higher (52.4  3.3) in Au-C225-Cy3 treated cells
`as compared to C225-Cy3 (36.0  2.6) with a p value of <0.05,
`suggesting increased accumulation of Au-C225-Cy3 in the recy-
`cling compartment, corroborating faster endocytosis of the recep-
`tor (Table 1, Fig. 2C). No difference in lysosomal colocalization
`was observed (Fig. S4). It was also not previously known whether
`any antibody-internalized receptor was trafficked to the lysosome
`(21, 23). Here, we demonstrate that a distinct amount of cetux-
`imab induced internalized EGFR is trafficked to the lysosomes.
`Similar results were obtained in PANC-1 cells, Au-C225-Cy3
`promoted significant higher localization (41.5  4.8) to EEA
`as compared to C225-Cy3 (24.1  3.5) (p < 0.05) (Table 1,
`Fig. 2B, Figs. S4, S5, S6, and S7). Since, C225 alone did not in-
`duce significant EGFR endocytosis in AsPC-1 cells at 1 h; we did
`not quantify the colocalization in this case. However, Au-C225-
`Cy3 treated AsPC-1 cells demonstrated notable localization of
`EGFR to early endosomes, Golgi complex, transferrin, and lyso-
`somes (Table 1, Figs. S4, S5, S6, and S7). Since gold nanoparticles
`in the Au-C225 conjugate cannot be documented by confocal
`microscopy, transmission electron microscopy (TEM) was per-
`formed.
`Internalization of gold nanoparticles
`in PANC-1,
`AsPC-1, and MiaPaca2 cells in double layered-membrane bound
`vesicles is demonstrated (Fig. 2 A–C, right, respectively). These
`data suggest that conjugation of C225 to gold nanoparticles could
`
`Fig. 1. Conjugation with gold nanoparticle accelerates faster endocytosis of
`EGFR receptors by C225. Fluorescence image representing the binding of
`C225-Cy3 and Au-C225-Cy3 to EGFR in PANC-1, AsPC-1, and MiaPaca2 human
`pancreatic cancer cells. The nucleus is stained with DAPI (blue). Cells were
`treated separately with C225-Cy3 and Au-C225-Cy3 for different time points
`starting from 5 min–1 h at 37 °C. A significantly higher internalization of
`EGFR was observed in PANC-1 (1 h), AsPC-1 (1 h) and MiaPaca2 (1 h) cells
`A–C right, respectively) with Au-C225-Cy3 treatment as compared to C225-
`Cy3 alone (A–C left, respectively). All other time points are presented in
`Fig. S1-PANC-1, Fig. S2-AsPC-1, Fig. S3-MiaPaca2).
`
`C225 for 30 min inhibited the uptake of Au-C225, indicating
`EGFR mediated uptake of Au-C225 by these cells (22). A de-
`tailed time course of C225-Cy3 and Au-C225-Cy3 induced EGFR
`internalization of the cell lines described above is provided in
`Fig. S1-PANC-1, Fig. S2-ASPC-1, Fig. S3 MiaPaca2. It is also im-
`portant to note here that the information regarding endocytic
`sorting of antibody-receptor complex and the mechanism of en-
`docytosis is still lacking (21, 23). We are demonstrating in this
`paper the mechanism and endocytic sorting of antibody-receptor
`complex in nanoconjugated and nonconjugated form. It is also
`
`Table 1. Quantification of colocalization in different organelles
`
`Organelles
`
`Early endosome
`
`Golgi
`
`Transferrin
`
`Lysosome
`
`Cell Types
`
`C225
`
`Au-C225
`
`C225
`
`Au-C225
`
`C225
`
`Au-C225
`
`C225
`
`Au-C225
`
`PANC-1
`24.1 ± 3.5
`AsPC-1
`ND
`MiaPaca2
`45.5 ± 4.9
`ND ¼ Not Done
`*p < 0.05
`
`41.5 ± 4.8*
`15.2 ± 1.3
`47.1 ± 2.8
`
`23.1 ± 2.6
`ND
`12.5 ± 1.6
`
`21.6 ± 1.8
`38.4 ± 3.3
`24.2 ± 2.1*
`
`35.8 ± 0.7
`ND
`36.0 ± 2.6
`
`36.6 ± 2.6
`46.9 ± 1.4
`52.4 ± 3.3*
`
`32.1 ± 3.2
`ND
`25.0 ± 2.8
`
`37.7 ± 3.6
`49.9 ± 2.3
`29.9 ± 1.8
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`Scheme 1. Role of dynamin in EGFR endocytosis; highlights a possible role
`of dynamin in the formation of membrane constriction in EGFR endocytosis
`induced either by C225 or Au-C225.
`
`Since C225-Cy3 induced only a marginal induction of EGFR
`endocytosis in AsPC-1 cells at 1 h, we extended the incubation
`time and treated AsPC-1 cells with either C225-Cy3 or Au-C225-
`Cy3 for 2 h to determine the role of dynamin. Expression of either
`
`ENGINEERING
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`APPLIEDBIOLOGICAL
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`SCIENCES
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`Fig. 3. Role of dynamin in C225-Cy3 induced internalization of EGFR. Figure
`demonstrates the role of dynamin in C225-Cy3 induced internalization of
`EGFR. Different human pancreatic cancer cells were infected with adenovirus
`that expresses either the WT or the K44A GTPase dynamin mutant followed
`by treatment with C225-Cy3 for 1 h. Internalization was monitored using con-
`focal microscopy. A–C demonstrate the role of dynamin in C225-Cy3 induced
`internalization of EGFR in PANC-1, AsPC-1, and MiaPaca2 cells, respectively.
`
`Fig. 2. C225-Cy3 and Au-C225-Cy3-induced endocytosis of EGFR in different
`compartments. Figure demonstrates colocalization of C225-Cy3 (left) and
`Au-C225-Cy3 (right) in different compartments. Cells were incubated with
`either C225-Cy3 or Au-C225-Cy3 for 1 h followed by colocalization with
`different reagents. A and C demonstrate colocalization of C225-Cy3 and
`Au-C225-Cy3 to the Golgi complex and transferrin in the case of MiaPaca2
`cells. B demonstrates localization to EEA when PANC-1 cells were treated
`with C225-Cy3 and Au-C225-Cy-3 for 1 h.
`Internalization of gold nano-
`particles
`in PANC-1, AsPC-1, and MiaPaca2 cells
`in double layered-
`membrane bound vesicles is observed by TEM and demonstrated in A–C,
`right, respectively.
`
`enhance the internalization of EGFR in both metastatic and pri-
`mary cancer cells. Colocalization with the transferrin compart-
`ment suggests involvement of the receptor mediated endocytic
`pathway by conjugation of antibody with gold nanoparticles
`(25, 26). A high degree of compartmentalization of receptor in
`specific organelles suggests the possibility of surface engineering
`of nanoparticle for specific intracellular targeting.
`
`Conjugation to Gold Nanoparticles Altered the Mechanism of C225
`Induced Endocytosis of EGFR. To determine if nanoconjugation
`modulated the mechanism of C225 induced endocytosis of
`EGFR, we tested the role of dyn-2 in this process. Dyn-2 is a sig-
`nal transducing GTPase that has been implicated in EGF-induced
`endocytosis of EGFR (26, 27). Accumulating evidence suggests
`the significant involvement of dyn-2 in generation, constriction,
`membrane ruffling, and fission of endocytotic vesicle stalks and is
`involved in both clathrin-dependent and independent pathways
`(Scheme 1) (28). However, the role of dyn-2 in C225 induced en-
`docytosis of EGFR has not been elucidated. To test the involve-
`ment of dyn-2 in C225 induced endocytosis of EGFR, wild-type
`or mutant dyn-2 (K44A lacking GTPase activity) were expressed
`in PANC-1, AsPC-1, and MiaPaca2 cells respectively, followed
`by treatment with C225-Cy3 or Au-C225-Cy3. Expression of
`WTor K44A dyn-2 mutant was confirmed by western blot analysis
`(Fig. S8). In PANC-1 cells, expression of mutant dyn-2 inhibited
`C225-Cy3 induced endocytosis of EGFR (evident from the per-
`sistent Cy3 florescence at the membrane) as compared to PANC-
`1 cells expressing WT dyn-2. However, upon nanoconjugation
`(Au-C225-Cy3), enhanced endocytosis was observed in the same
`cells despite expression of mutant dyn-2. However, in MiaPaca2
`cells expression of mutant dyn-2 inhibited both C225-Cy3 and
`Au-C225-Cy3 induced endocytosis of EGFR.
`
`Bhattacharyya et al.
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`PNAS ∣ August 17, 2010 ∣
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`vol. 107 ∣ no. 33 ∣ 14543
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`Thus, the regulation of dynamin in EGFR endocytosis upon
`nanoconjugation may provide valuable insight in to the mecha-
`nism of C225 function.
`
`Discussion
`Cells use endocytosis as a tool to internalize a variety of molecules
`(cargo) to communicate with the extracellular environments.
`These endocytic processes are extremely complex and tightly
`regulated (29). Cetuximab is approved by the FDA for the treat-
`ment of EGFR-positive colorectal cancer. However, the mecha-
`nism of communication between cetuximab and EGFR expres-
`sing cancer cells has not been elucidated. Furthermore, it has also
`been unknown whether nanoconjugation could lead to the alter-
`nation of the mechanism of endocytosis of a targeting agent.
`Here, we demonstrate that nanoconjugation plays an important
`role in regulating the mechanism of C225; it uniformly promotes
`faster EGFR endocytosis across different pancreatic cancer cell
`lines of metastatic and primary origin. This finding is of clinical
`significance since attenuation of membrane presentation of the
`receptor implies reduced signaling through its corresponding
`ligand. Furthermore, the pattern of EGFR endocytosis is distinct
`in metastatic vs. primary cell lines. Whether such a distinct pat-
`terning of EGFR in highly aggressive and metastatic cell line is
`responsible for metastatic potential is not known but this is an
`important finding that requires further investigation.
`It is also important to understand whether nanoconjugation
`alters the mechanism and hence compartmentalization of anti-
`body induced internalization of EGFR and their subsequent
`functional consequences. Current studies have speculated the
`involvement of clathrin- and caveolin-dependent vesicle forma-
`tion in nanoparticle mediated cellular endocytosis (30). It has
`been reported that size is one of the key factors that determine
`the cellular uptake of the nano-composites (5). However, the up-
`stream event of nanoparticle entry to the cellular compartment
`and the molecular machinery that drives the dynamics of the in-
`ternalization to cancer cells is not known. Evidence suggests an
`important role of dyn-2 in many cellular processes such as EGFR
`and other receptor endocytosis (28, 29, 31). EGF stimulated
`EGFRs have been found to involve both clathrin-dependent
`and independent pathways (32). In our study, we have addressed
`whether dyn-2, implicated in both clathrin mediated endocytosis
`(CME) and clathrin independent endocytosis (CI), is involved in
`nanoconjugated-C225 induced EGFR endocytosis in pancreatic
`cancer cells. Furthermore, dynamin is also evolving as a new ther-
`apeutic target in various diseases. In both PANC-1 and MiaPaca2
`cells mutant dyn-2 significantly inhibited free C225 induced
`endocytosis of EGFR, suggesting the involvement of CME path-
`ways. However, nanoconjugation resulted in increased endo-
`cytosis of EGFR despite expression of mutant dyn-2, implicating
`a possible role of dyn-2 independent, CI pathways in nanoconju-
`gated form. These results suggest that C225 induced endocytosis
`of EGFR may require partial involvement of both the CI and
`CME pathways. Further investigation is required to substantiate
`the role of CI and dyn-2 independent pathways by probing other
`players such as CDC42, ARF, or RHO GTPase (29, 33, 34). On
`the other hand, in AsPC-1 cells, nanoconjugation altered the
`mechanism of C225 induced endocytosis of EGFR from dyn-2
`independent to dyn-2 dependent pathway, implicating a possible
`shift from CI to CME pathways. However, additional studies are
`required to identify other factors and molecular players involved
`during nanoparticle mediated receptor endocytosis. This under-
`standing will help us to engineer and design suitable nanomachi-
`nery to regulate intracellular pathways.
`In summary, we have demonstrated that membrane receptor
`endocytosis is distinct in metastatic vs. primary human pancreatic
`cancer cell lines. Nanoconjugation induces faster endocytosis
`of EGFR, influences the compartmentalization, and alters the
`mechanism of endocytosis. Thus, nanoconjugation cannot be
`
`Fig. 4. Role of dynamin in Au-C225-Cy3 induced internalization of EGFR.
`Figure demonstrates the role of dynamin in Au-C225-Cy3 induced inter-
`nalization of EGFR. Different pancreatic cancer cells were infected with
`adenovirus that expresses either the WT or the K44A GTPase dynamin mutant
`followed by treatment with Au-C225-Cy3 for 1 h.
`Internalization was
`monitored using confocal microscopy. A–C demonstrate the role of dynamin
`in Au-C225-Cy3 induced internalization of EGFR in PANC-1, AsPC-1, and
`MiaPaca2 cells, respectively.
`
`WT or mutant dyn-2 did not affect C225-induced endocytosis of
`EGFR, confirming that C225-induced EGFR endocytosis is dyn-
`2 independent in AsPC-1 cells. However, increased inhibition of
`EGFR endocytosis was observed when AsPC-1 cells, expressing
`mutant dyn-2 was treated with Au-C225-Cy3. These results
`clearly demonstrate the alteration of C225-induced endocytosis
`from dyn-2 independent to dyn-2 dependent pathway in this cell
`upon nanoconjugation. Therefore, in AsPC-1 cells characterized
`by their high metastatic potential, nanoconjugation shifted the
`mechanism of C225-Cy3 induced EGFR endocytosis from dyna-
`min independent to dynamin dependent pathway. These results
`clearly demonstrate that nanoconjugate specifically modulates
`the involvement of dyn-2 in pancreatic cancer cells with high
`EGFR expression. In PANC-1 cells with high EGFR expression,
`where C225-Cy3 induced EGFR endocytosis was dyn-2 depen-
`dent, switched it to dyn-2 independent pathway upon nanocon-
`jugation. On the otherhand, in AsPC-1 cells with similar level
`of EGFR expression as PANC-1 cells, where C225-Cy3 induced
`EGFR endocytosis was dyn-2 independent, switched it to dyn-2
`dependent pathways by Au-C225-Cy3. However, in MiaPaca2
`cells with low EGFR expression, both C225-Cy3 and Au-C225-
`Cy3 induced EGFR endocytosis remained dyn-2 dependent.
`
`14544 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1006507107
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`construed as an innocuous reaction involved in attaching the
`targeting agent to the nanoparticle, instead it may distinctly alter
`the cellular processes at the molecular level. The mechanism of
`such alteration of endocytic pathways upon nanoconjugation is
`unique and warrants further investigation. It is possible that
`nanoconjugation enables C225 to induce receptor clustering to
`a greater extent than the free form (35) which results in faster
`endocytosis leading to attenuation of membrane presentation
`of the receptor. These findings provide the mechanism through
`which nanoconjugation alters C225 function and may have signif-
`icant clinical implications in the treatment of EGFR-positive hu-
`man cancers. Furthermore, this finding will also help to expedite
`the design of nanofabrication for efficient targeting and therapy.
`
`Methods
`Reagents. Antibody was principally purchased from Cell Signaling and Santa
`Cruz, EEA1 (Cell Signaling, cat #2411), Dynamin (Santa Cruiz, sc-11362)
`Lysotracker (Invitrogen, L7526), Golgi antibody (TNF46, Sigma), Cy3 labeling
`kit (GE healthcare, PA33001), Cetuximab (Bristol Mayer Squib), Transferrin
`antibody with Alex Flour 488, Para formaldehyde (Electron Microscopy
`Science, 15710), BSA (Fischer Scientific, S-5058), and ammonium chloride
`(Sigma, A0171). Mutant (K44A) and WT dyn-2 adenovirus was a kind gift
`from Mark McNiven, Mayo Clinic.
`
`Cell Culture. The pancreatic cancer cell lines AsPC-1, PANC-1, and MiaPaca-2
`were purchased from American Type Culture Collection and cultured using
`RPMI medium 1640 and DMEM with L-glutamine (Cellgro Mediatech, Inc.)
`supplemented with 10% FBS and 1% antibiotics (penicillin-streptomycin).
`
`Synthesis of Gold Nanoparticles. Gold nanoparticles (AuNPs) were synthesized
`from tetrachlorauric acid by wet chemical methods using sodium boro-
`hydride as a reducing agent as previously described in ref. 36. Briefly, an
`aqueous stock solution of tetrachloroauric acid (HAuCl4) was reduced with
`an aqueous solution of sodium borohydride (NaBH4) under vigorous stirring
`(18). Stirring continued for 12 h to obtain AuNPs used in this study. AuNPs
`thus formed were characterized using UV-Vis and TEM confirming ∼5 nm size
`spherical nanoparticles formed by this method.
`
`Synthesis and Characterization of Gold Nanoconjugates Containing Anti-EGFR
`Antibody Labeled with Cy3 dye. The monoclonal antibody C225 was conju-
`gated with monoclonal antibody labeling dye Cy3 according to manufac-
`turers protocol (GE healthcare, PA33001). In brief, the antibody (2 mg∕mL)
`was diluted (1∶1) with PBS buffer and then incubated with Cy3 dye for
`30 min in the presence of coupling buffer. Free dye was separated through
`a gel filtration column provided in the kit. Cy3-labeled C225 (C225-Cy3) thus
`obtained was used to synthesize Au-C225-Cy3 by a simple mixing technique
`by adding 2 μg∕mL of C225-Cy3 to AuNP solution for 1 h. Au-C225-Cy3 was
`
`separated from unbound C225-Cy3 by ultracentrifugation as previously
`described in ref. 18. The ultracentrifugation was repeated twice after
`washing and the loose pellet resuspended in distilled water for use (see
`legend of Fig. S8).
`
`Immunofluorescence Microscopy. Cells (2 × 104∕well) were seeded in four well
`chamber slides. After 24 h, each well was treated either with C225-Cy3 or Au-
`C225-Cy3 in a concentration of 2 μg∕mL at 37 °C. Endocytosis was followed
`over time (5 min–1 h) by confocal microscopy. In separate experiments cells
`were also incubated with C225-Cy3 and Au-C225-Cy3 in cold for 30 min. At
`the end of the experiments each well in the chamber slide was washed with
`cold PBS (three times). Cells were fixed in 2% paraformaldehyde in PBS at
`room temperature for 15 min. At the end of fixation wells were washed with
`PBS and then mounted in a mounting media containing the nuclear stain
`DAPI (Vectashield). For colocalization experiments, cells were fixed and then
`blocked with 2% BSA in PBS for 2 h followed by incubation with respective
`antibodies with appropriate dilution as suggested in the company manual.
`After incubation at 4 °C, cells were washed three times with PBS. To detect
`the primary antibody that bind specifically to organelles (Golgi or early
`endosome), a secondary antibody of goat anti-rabbit IgG1 conjugated with
`Alexa fluor 488 were added to the chamber. Pictures were taken at
`63X∕1.2 W with scan zoom 1.0 and 2.0.
`To determine the role of dynamin, cells were seeded in four well chamber
`slides at 2 × 104 per well per mL. The next day cells were infected with
`adenovirus that expresses either the WT or the K44A GTPase dynamin
`mutant. The medium was changed after 48 h and treated as above.
`
`Quantification of Compartmentalization. Image quantification was done using
`KS400 image analysis software (Carl Zeiss Micro Imaging). For quantification
`of colocalization a minimum threshold of red and green channels was
`selected. Images were analyzed from different areas of the plate and percen-
`tage of colocalization was calculated from the amount of colocalized areas
`from the total green or red area. The data represent the calculation of the
`snaps taken from five different areas (n ¼ 5) and having at least 20–40
`number of cells with data presented as mean  SEM.
`
`Transmission Electron Microscopy. Pancreatic cancer cells were seeded and
`grown in 100 mm tissue culture plates. At ∼70% confluence, cells were
`treated with Au-C225 (Au∶C225 ∼ 0.5) for 2 h followed by thorough washing
`of the cells with 1X PBS and then added to Trumps fixative for microtome
`sectioning for TEM microscopy as previously reported in ref. 37.
`
`Statistical Analysis. Statistical analysis was done by a two-tailed student t-test
`and P < 0.05 is considered as significant.
`
`ACKNOWLEDGMENTS. Supported by National Institutes of Health (NIH) Grant
`CA135011, CA136494 and University of Texas MD Anderson Cancer Grant
`UTMD-1 (to P.M.).
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`Bhattacharyya et al.
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`PNAS ∣ August 17, 2010 ∣
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`vol. 107 ∣ no. 33 ∣ 14545
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